Molecules for disease detection and treatment

ABSTRACT

The present invention provides purified disease detection and treatment molecule polynucleotides (mddt). Also encompassed are the polypeptides (MDDT) encoded by mddt. The invention also provides for the use of mddt, or complements, oligonucleotides, or fragments thereof in diagnostic assays. The invention further provides for vectors and host cells containing mddt for the expression of MDDT. The invention additionally provides for the use of isolated and purified MDDT to induce anitbodies and to screen libraries of compounds and the use of anti-MDDT antibodies in diagnostic assays. Also provided are microarrays containing mddt and methods of use.

TECHNICAL FIELD

The present invention relates to molecules for disease detection andtreatment and to the use of these sequences in the diagnosis, study,prevention, and treatment of diseases associated with, as well aseffects of exogenous compounds on, the expression of molecules fordisease detection and treatment.

BACKGROUND OF THE INVENTION

The human genome is comprised of thousands of genes, many encoding geneproducts that function in the maintenance and growth of the variouscells and tissues in the body. Aberrant expression or mutations in thesegenes and their products is the cause of, or is associated with, avariety of human diseases such as cancer and other cell proliferativedisorders. The identification of these genes and their products is thebasis of an ever-expanding effort to find markers for early detection ofdiseases, and targets for their prevention and treatment.

For example, cancer represents a type of cell proliferative disorderthat affects nearly every tissue in the body. A wide variety ofmolecules, either aberrantly expressed or mutated, can be the cause of,or involved with, various cancers because tissue growth involves complexand ordered patterns of cell proliferation, cell differentiation, andapoptosis. Cell proliferation must be regulated to maintain both thenumber of cells and their spatial organization. This regulation dependsupon the appropriate expression of proteins which control cell cycleprogression in response to extracellular signals such as growth factorsand other mitogens, and intracellular cues such as DNA damage ornutrient starvation. Molecules which directly or indirectly modulatecell cycle progression fall into several categories, including growthfactors and their receptors, second messenger and signal transductionproteins, oncogene products, tumor-suppressor proteins, andmitosis-promoting factors. Aberrant expression or mutations in any ofthese gene products can result in cell proliferative disorders such ascancer. Oncogenes are genes generally derived from normal genes that,through abnormal expression or mutation, can effect the transformationof a normal cell to a malignant one (oncogenesis). Oncoproteins, encodedby oncogenes, can affect cell proliferation in a variety of ways andinclude growth factors, growth factor receptors, intracellular signaltransducers, nuclear transcription factors, and cell-cycle controlproteins. In contrast, tumor-suppressor genes are involved in inhibitingcell proliferation. Mutations which cause reduced or loss of function intumor-suppressor genes result in aberrant cell proliferation and cancer.Thus a wide variety of genes and their products have been found that areassociated with cell proliferative disorders such as cancer, but manymore may exist that are yet to be discovered.

DNA-based arrays can provide a simple way to explore the expression of asingle polymorphic gene or a large number of genes. When the expressionof a single gene is explored, DNA-based arrays are employed to detectthe expression of specific gene variants. For example, a p53 tumorsuppressor gene array is used to determine whether individuals arecarrying mutations that predispose them to cancer. A cytochrome p450gene array is useful to determine whether individuals have one of anumber of specific mutations that could result in increased drugmetabolism, drug resistance or drug toxicity.

DNA-based array technology is especially relevant for the rapidscreening of expression of a large number of genes. There is a growingawareness that gene expression is affected in a global fashion. Agenetic predisposition, disease or therapeutic treatment may affect,directly or indirectly, the expression of a large number of genes. Insome cases the interactions may be expected, such as when the genes arepart of the same signaling pathway. In other cases, such as when thegenes participate in separate signaling pathways, the interactions maybe totally unexpected. Therefore, DNA-based arrays can be used toinvestigate how genetic predisposition, disease, or therapeutictreatment affects the expression of a large number of genes.

The discovery of new molecules for disease detection and treatmentsatisfies a need in the art by providing new compositions which areuseful in the diagnosis, study, prevention, and treatment of diseasesassociated with, as well as effects of exogenous compounds on, theexpression of molecules for disease detection and treatment

SUMMARY OF THE INVENTION

The present invention relates to human disease detection and treatmentmolecule polynucleotides (mddt) as presented in the Sequence Listing.The mddt uniquely identify genes encoding structural, functional, andregulatory disease detection and treatment molecules.

The invention provides an isolated polynucleotide comprising apolynucleotide sequence selected from the group consisting of a) apolynucleotide sequence selected from the group consisting of SEQ IDNO:1-45; b) a naturally occurring polynucleotide sequence having atleast 90% sequence identity to a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:1-45; c) a polynucleotide sequencecomplementary to a); d) a polynucleotide sequence complementary to b);and e) an RNA equivalent of a) through d). In one alternative, thepolynucleotide comprises a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:1-45. In another alternative, thepolynucleotide comprises at least 60 contiguous nucleotides of apolynucleotide sequence selected from the group consisting of a) apolynucleotide sequence selected from the group consisting of SEQ IDNO:1-45; b) a naturally occurring polynucleotide sequence having atleast 90% sequence identity to a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:1-45; c) a polynucleotide sequencecomplementary to a); d) a polynucleotide sequence complementary to b);and e) an RNA equivalent of a) through d). The invention furtherprovides a composition for the detection of expression of diseasedetection and treatment molecule polynucleotides comprising at least oneisolated polynucleotide comprising a polynucleotide sequence selectedfrom the group consisting of a) a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:1-45; b) a naturally occurringpolynucleotide sequence having at least 90% sequence identity to apolynucleotide sequence selected from the group consisting of SEQ IDNO:1-45; c) a polynucleotide sequence complementary to a); d) apolynucleotide sequence complementary to b); and e) an RNA equivalent ofa) through d); and a detectable label.

The invention also provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide comprising apolynucleotide sequence selected from the group consisting of a) apolynucleotide sequence selected from the group consisting of SEQ IDNO:1-45; b) a naturally occurring polynucleotide sequence having atleast 90% sequence identity to a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:1-45; c) a polynucleotide sequencecomplementary to a); d) a polynucleotide sequence complementary to b);and e) an RNA equivalent of a) through d). The method comprises a)amplifying said target polynucleotide or a fragment thereof usingpolymerase chain reaction amplification, and b) detecting the presenceor absence of said amplified target polynucleotide or fragment thereof,and, optionally, if present, the amount thereof.

The invention also provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide comprising apolynucleotide sequence selected from the group consisting of a) apolynucleotide sequence selected from the group consisting of SEQ IDNO:1-45; b) a naturally occurring polynucleotide sequence having atleast 90% sequence identity to a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:1-45; c) a polynucleotide sequencecomplementary to a); d) a polynucleotide sequence complementary to b);and e) an RNA equivalent of a) through d). The method comprises a)hybridizing the sample with a probe comprising at least 20 contiguousnucleotides comprising a sequence complementary to said targetpolynucleotide in the sample, and which probe specifically hybridizes tosaid target polynucleotide, under conditions whereby a hybridizationcomplex is formed between said probe and said target polynucleotide, andb) detecting the presence or absence of said hybridization complex, and,optionally, if present, the amount thereof. In one alternative, theprobe comprises at least 30 contiguous nucleotides. In anotheralternative, the probe comprises at least 60 contiguous nucleotides.

The invention further provides a recombinant polynucleotide comprising apromoter sequence operably linked to an isolated polynucleotidecomprising a polynucleotide sequence selected from the group consistingof a) a polynucleotide sequence selected from the group consisting ofSEQ ID NO:1-45; b) a naturally occurring polynucleotide sequence havingat least 90% sequence identity to a polynucleotide sequence selectedfrom the group consisting of SEQ ID NO:1-45; c) a polynucleotidesequence complementary to a); d) a polynucleotide sequence complementaryto b); and e) an RNA equivalent of a) through d). In one alternative,the invention provides a cell transformed with the recombinantpolynucleotide. In another alternative, the invention provides atransgenic organism comprising the recombinant polynucleotide. In afurther alternative, the invention provides a method for producing adisease detection and treatment molecule polypeptide, the methodcomprising a) culturing a cell under conditions suitable for expressionof the disease detection and treatment molecule polypeptide, whereinsaid cell is transformed with the recombinant polynucleotide, and b)recovering the disease detection and treatment molecule polypeptide soexpressed.

The invention also provides a purified disease detection and treatmentmolecule polypeptide (MDDT) encoded by at least one polynucleotidecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NO:1-45. Additionally, the invention provides an isolatedantibody which specifically binds to the disease detection and treatmentmolecule polypeptide. The invention further provides a method ofidentifying a test compound which specifically binds to the diseasedetection and treatment molecule polypeptide, the method comprising thesteps of a) providing a test compound; b) combining the diseasedetection and treatment molecule polypeptide with the test compound fora sufficient time and under suitable conditions for binding; and c)detecting binding of the disease detection and treatment moleculepolypeptide to the test compound, thereby identifying the test compoundwhich specifically binds the disease detection and treatment moleculepolypeptide.

The invention further provides a microarray wherein at least one elementof the microarray is an isolated polynucleotide comprising at least 60contiguous nucleotides of a polynucleotide comprising a polynucleotidesequence selected from the group consisting of a) a polynucleotidesequence selected from the group consisting of SEQ ID NO:1-45; b) anaturally occurring polynucleotide sequence having at least 90% sequenceidentity to a polynucleotide sequence selected from the group consistingof SEQ ID NO:1-45; c) a polynucleotide sequence complementary to a); d)a polynucleotide sequence complementary to b); and e) an RNA equivalentof a) through d). The invention also provides a method for generating atranscript image of a sample which contains polynucleotides. The methodcomprises a) labeling the polynucleotides of the sample, b) contactingthe elements of the microarray with the labeled polynucleotides of thesample under conditions suitable for the formation of a hybridizationcomplex, and c) quantifying the expression of the polynucleotides in thesample.

Additionally, the invention provides a method for screening a compoundfor effectiveness in altering expression of a target polynucleotide,wherein said target polynucleotide comprises a polynucleotide sequenceselected from the group consisting of a) a polynucleotide sequenceselected from the group consisting of SEQ ID NO:1-45; b) a naturallyoccurring polynucleotide sequence having at least 90% sequence identityto a polynucleotide sequence selected from the group consisting of SEQID NO:1-45; c) a polynucleotide sequence complementary to a); d) apolynucleotide sequence complementary to b); and e) an RNA equivalent ofa) through d). The method comprises a) exposing a sample comprising thetarget polynucleotide to a compound, and b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.

The invention further provides a method for assessing toxicity of a testcompound, said method comprising a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide comprising apolynucleotide sequence selected from the group consisting of i) apolynucleotide sequence selected from the group consisting of SEQ IDNO:1-45; ii) a naturally occurring polynucleotide sequence having atleast 90% sequence identity to a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:1-45; iii) a polynucleotide sequencecomplementary to i), iv) a polynucleotide sequence complementary to ii),and v) an RNA equivalent of i)-iv). Hybridization occurs underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence selected fromthe group consisting of i) a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:1-45; ii) a naturally occurringpolynucleotide sequence having at least 90% sequence identity to apolynucleotide sequence selected from the group consisting of SEQ IDNO:1-45; iii) a polynucleotide sequence complementary to i), iv) apolynucleotide sequence complementary to ii), and v) an RNA equivalentof i)-iv), and alternatively, the target polynucleotide comprises afragment of a polynucleotide sequence selected from the group consistingof i)-v) above; c) quantifying the amount of hybridization complex; andd) comparing the amount of hybridization complex in the treatedbiological sample with the amount of hybridization complex in anuntreated biological sample, wherein a difference in the amount ofhybridization complex in the treated biological sample is indicative oftoxicity of the test compound.

The invention further provides an isolated polypeptide comprising anamino acid sequence selected from the group consisting of a) an aminoacid sequence selected from the group consisting of SEQ ID NO:46-90, b)a naturally occurring amino acid sequence having at least 90% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NO:46-90, c) a biologically active fragment of an amino acidsequence selected from the group consisting of SEQ ID NO:46-90, and d)an immunogenic fragment of an amino acid sequence selected from thegroup consisting of SEQ ID NO:46-90. In one alternative, the inventionprovides an isolated polypeptide comprising the amino acid sequence ofSEQ ID NO:46-90.

DESCRIPTION OF THE TABLES

Table 1 shows the sequence identification numbers (SEQ ID NO:s) andtemplate identification numbers (template IDs) corresponding to thepolynucleotides of the present invention, along with their GenBank hits(GI Numbers), probability scores, and functional annotationscorresponding to the GenBank hits.

Table 2 shows the sequence identification numbers (SEQ ID NO:s) andtemplate identification numbers (template IDs) corresponding to thepolynucleotides of the present invention, along with polynucleotidesegments of each template sequence as defined by the indicated “start”and “stop” nucleotide positions. The reading frames of thepolynucleotide segments and the Pfam hits, Pfam descriptions, andE-values corresponding to the polypeptide domains encoded by thepolynucleotide segments are indicated.

Table 3 shows the sequence identification numbers (SEQ ID NO:s) andtemplate identification numbers (template IDs) corresponding to thepolynucleotides of the present invention, along with polynucleotidesegments of each template sequence as defined by the indicated “start”and “stop” nucleotide positions. The reading frames of thepolynucleotide segments are shown, and the polypeptides encoded by thepolynucleotide segments constitute either signal peptide (SP) ortransmembrane (TM) domains, as indicated. The membrane topology of theencoded polypeptide sequence is indicated, the N-terminus (N) listed asbeing oriented to either the cytosolic (in) or non-cytosolic (out) sideof the cell membrane or organelle.

Table 4 shows the sequence identification numbers (SEQ ID NO:s)corresponding to the polynucleotides of the present invention, alongwith component sequence identification numbers (component IDs)corresponding to each template. The component sequences, which were usedto assemble the template sequences, are defined by the indicated “start”and “stop” nucleotide positions along each template.

Table 5 shows the tissue distribution profiles for the templates of theinvention.

Table 6 shows the sequence identification numbers (SEQ ID NO:s)corresponding to the polypeptides of the present invention, along withthe reading frames used to obtain the polypeptide segments, the lengthsof the polypeptide segments, the “start” and “stop” nucleotide positionsof the polynucleotide sequences used to define the encoded polypeptidesegments, the GenBank hits (GI Numbers), probability scores, andfunctional annotations corresponding to the GenBank hits.

Table 7 summarizes the bioinformatics tools which are useful foranalysis of the polynucleotides of the present invention. The firstcolumn of Table 7 lists analytical tools, programs, and algorithms, thesecond column provides brief descriptions thereof, the third columnpresents appropriate references, all of which are incorporated byreference herein in their entirety, and the fourth column presents,where applicable, the scores, probability values, and other parametersused to evaluate the strength of a match between two sequences (thehigher the score, the greater the homology between two sequences).

DETAILED DESCRIPTION OF THE INVENTION

Before the nucleic acid sequences and methods are presented, it is to beunderstood that this invention is not limited to the particularmachines, methods, and materials described. Although particularembodiments are described, machines, methods, and materials similar orequivalent to these embodiments may be used to practice the invention.The preferred machines, methods, and materials set forth are notintended to limit the scope of the invention which is limited only bythe appended claims.

The singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. All technical and scientificterms have the meanings commonly understood by one of ordinary skill inthe art. All publications are incorporated by reference for the purposeof describing and disclosing the cell lines, vectors, and methodologieswhich are presented and which might be used in connection with theinvention. Nothing in the specification is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

Definitions

As used herein, the lower case “mddt” refers to a nucleic acid sequence,while the upper case “MDDT” refers to an amino acid sequence encoded bymddt. A “full-length” mddt refers to a nucleic acid sequence containingthe entire coding region of a gene endogenously expressed in humantissue.

“Adjuvants” are materials such as Freund's adjuvant, mineral gels(aluminum hydroxide), and surface active substances (lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, and dinitrophenol) which may be administered to increase ahost's immunological response.

“Allele” refers to an alternative form of a nucleic acid sequence.Alleles result from a “mutation,” a change or an alternative reading ofthe genetic code. Any given gene may have none, one, or many allelicforms. Mutations which give rise to alleles include deletions,additions, or substitutions of nucleotides. Each of these changes mayoccur alone, or in combination with the others, one or more times in agiven nucleic acid sequence. The present invention encompasses allelicmddt.

“Amino acid sequence” refers to a peptide, a polypeptide, or a proteinof either natural or synthetic origin. The amino acid sequence is notlimited to the complete, endogenous amino acid sequence and may be afragment, epitope, variant, or derivative of a protein expressed by anucleic acid sequence.

“Amplification” refers to the production of additional copies of asequence and is carried out using polymerase chain reaction (PCR)technologies well known in the art.

“Antibody” refers to intact molecules as well as to fragments thereof,such as Fab, F(ab′)₂, and Fv fragments, which are capable of binding theepitopic determinant. Antibodies that bind MDDT polypeptides can beprepared using intact polypeptides or using fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide orpeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)can be derived from the translation of RNA, or synthesized chemically,and can be conjugated to a carrier protein if desired. Commonly usedcarriers that are chemically coupled to peptides include bovine serumalbumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupledpeptide is then used to immunize the animal.

“Antisense sequence” refers to a sequence capable of specificallyhybridizing to a target sequence. The antisense sequence may includeDNA, RNA, or any nucleic acid mimic or analog such as peptide nucleicacid (PNA); oligonucleotides having modified backbone linkages such asphosphorothioates, methylphosphonates, or benzylphosphonates;oligonucleotides having modified sugar groups such as 2′-methoxyethylsugars or 2′-methoxyethoxy sugars; or oligonucleotides having modifiedbases such as 5-methyl cytosine, 2′-deoxyuracil, or7-deaza-2′-deoxyguanosine.

“Antisense sequence” refers to a sequence capable of specificallyhybridizing to a target sequence. The antisense sequence can be DNA,RNA, or any nucleic acid mimic or analog.

“Antisense technology” refers to any technology which relies on thespecific hybridization of an antisense sequence to a target sequence.

A “bin” is a portion of computer memory space used by a computer programfor storage of data, and bounded in such a manner that data stored in abin may be retrieved by the program.

“Biologically active” refers to an amino acid sequence having astructural, regulatory, or biochemical function of a naturally occurringamino acid sequence.

“Clone joining” is a process for combining gene bins based upon thebins' containing sequence information from the same clone. The sequencesmay assemble into a primary gene transcript as well as one or moresplice variants.

“Complementary” describes the relationship between two single-strandednucleic acid sequences that anneal by base-pairing (5′-A-G-T-3′ pairswith its complement 3′-T-C-A-5′).

A “component sequence” is a nucleic acid sequence selected by a computerprogram such as PHRED and used to assemble a consensus or templatesequence from one or more component sequences.

A “consensus sequence” or “template sequence” is a nucleic acid sequencewhich has been assembled from overlapping sequences, using a computerprogram for fragment assembly such as the GELVIEW fragment assemblysystem (Genetics Computer Group (GCG), Madison, Wis.) or using arelational database management system (RDMS).

“Conservative amino acid substitutions” are those substitutions that,when made, least interfere with the properties of the original protein,i.e., the structure and especially the function of the protein isconserved and not significantly changed by such substitutions. The tablebelow shows amino acids which may be substituted for an original aminoacid in a protein and which are regarded as conservative substitutions.Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys AsnAsp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln,His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg,Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser,Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as abeta sheet or alpha helical conformation, (b) the charge orhydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain.

“Deletion” refers to a change in either a nucleic or amino acid sequencein which at least one nucleotide or amino acid residue, respectively, isabsent.

“Derivative” refers to the chemical modification of a nucleic acidsequence, such as by replacement of hydrogen by an alkyl, acyl, amino,hydroxyl, or other group.

The terms “element” and “array element” refer to a polynucleotide,polypeptide, or other chemical compound having a unique and definedposition on a microarray.

“E-value” refers to the statistical probability that a match between twosequences occurred by chance.

A “fragment” is a unique portion of mddt or MDDT which is identical insequence to but shorter in length than the parent sequence. A fragmentmay comprise up to the entire length of the defined sequence, minus onenucleotide/amino acid residue. For example, a fragment may comprise from10 to 1000 contiguous amino acid residues or nucleotides. A fragmentused as a probe, primer, antigen, therapeutic molecule, or for otherpurposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75,100, 150, 250 or at least 500 contiguous amino acid residues ornucleotides in length. Fragments may be preferentially selected fromcertain regions of a molecule. For example, a polypeptide fragment maycomprise a certain length of contiguous amino acids selected from thefirst 250 or 500 amino acids (or first 25% or 50%) of a polypeptide asshown in a certain defined sequence. Clearly these lengths areexemplary, and any length that is supported by the specification,including the Sequence Listing and the figures, may be encompassed bythe present embodiments.

A fragment of mddt comprises a region of unique polynucleotide sequencethat specifically identifies mddt, for example, as distinct from anyother sequence in the same genome. A fragment of mddt is useful, forexample, in hybridization and amplification technologies and inanalogous methods that distinguish mddt from related polynucleotidesequences. The precise length of a fragment of mddt and the region ofmddt to which the fragment corresponds are routinely determinable by oneof ordinary skill in the art based on the intended purpose for thefragment.

A fragment of MDDT is encoded by a fragment of mddt. A fragment of MDDTcomprises a region of unique amino acid sequence that specificallyidentifies MDDT. For example, a fragment of MDDT is useful as animmunogenic peptide for the development of antibodies that specificallyrecognize MDDT. The precise length of a fragment of MDDT and the regionof MDDT to which the fragment corresponds are routinely deteminable byone of ordinary skill in the art based on the intended purpose for thefragment.

A “full length” nucleotide sequence is one containing at least a startsite for translation to a protein sequence, followed by an open readingframe and a stop site, and encoding a “full length” polypeptide.

“Hit” refers to a sequence whose annotation will be used to describe agiven template. Criteria for selecting the top hit are as follows: ifthe template has one or more exact nucleic acid matches, the top hit isthe exact match with highest percent identity. If the template has noexact matches but has significant protein hits, the top hit is theprotein hit with the lowest E-value. If the template has no significantprotein hits, but does have significant non-exact nucleotide hits, thetop hit is the nucleotide hit with the lowest E-value.

“Homology” refers to sequence similarity either between a referencenucleic acid sequence and at least a fragment of an mddt or between areference amino acid sequence and a fragment of an MDDT.

“Hybridization” refers to the process by which a strand of nucleotidesanneals with a complementary strand through base pairing. Specifichybridization is an indication that two nucleic acid sequences share ahigh degree of identity. Specific hybridization complexes form underdefined annealing conditions, and remain hybridized after the “washing”step. The defined hybridization conditions include the annealingconditions and the washing step(s), the latter of which is particularlyimportant in determining the stringency of the hybridization process,with more stringent conditions allowing less non-specific binding, i.e.,binding between pairs of nucleic acid probes that are not perfectlymatched. Permissive conditions for annealing of nucleic acid sequencesare routinely determinable and may be consistent among hybridizationexperiments, whereas wash conditions may be varied among experiments toachieve the desired stringency.

Generally, stringency of hybridization is expressed with reference tothe temperature under which the wash step is carried out. Generally,such wash temperatures are selected to be about 5° C. to 20° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. An equation for calculatingT_(m) and conditions for nucleic acid hybridization is well known andcan be found in Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press, Plainview, N.Y.;specifically see volume 2, chapter 9.

High stringency conditions for hybridization between polynucleotides ofthe present invention include wash conditions of 68° C. in the presenceof about 0.2×SSC and about 0.1% SDS, for 1 hour. Alternatively,temperatures of about 65° C., 60° C., or 55° C. may be used. SSCconcentration may be varied from about 0.2 to 2×SSC, with SDS beingpresent at about 0.1%. Typically, blocking reagents are used to blocknon-specific hybridization. Such blocking reagents include, forinstance, denatured salmon sperm DNA at about 100-200 μg/ml. Usefulvariations on these conditions will be readily apparent to those skilledin the art. Hybridization, particularly under high stringencyconditions, may be suggestive of evolutionary similarity between thenucleotides. Such similarity is strongly indicative of a similar rolefor the nucleotides and their resultant proteins.

Other parameters, such as temperature, salt concentration, and detergentconcentration may be varied to achieve the desired stringency.Denaturants, such as formamide at a concentration of about 35-50% v/v,may also be used under particular circumstances, such as RNA:DNAhybridizations. Appropriate hybridization conditions are routinelydeterminable by one of ordinary skill in the art.

“Immunogenic” describes the potential for a natural, recombinant, orsynthetic peptide, epitope, polypeptide, or protein to induce antibodyproduction in appropriate animals, cells, or cell lines.

“Insertion” or “addition” refers to a change in either a nucleic oramino acid sequence in which at least one nucleotide or residue,respectively, is added to the sequence.

“Labeling” refers to the covalent or noncovalent joining of apolynucleotide, polypeptide, or antibody with a reporter moleculecapable of producing a detectable or measurable signal.

“Microarray” is any arrangement of nucleic acids, amino acids,antibodies, etc., on a substrate. The substrate may be a solid supportsuch as beads, glass, paper, nitrocellulose, nylon, or an appropriatemembrane.

“Linkers” are short stretches of nucleotide sequence which may be addedto a vector or an mddt to create restriction endonuclease sites tofacilitate cloning. “Polylinkers” are engineered to incorporate multiplerestriction enzyme sites and to provide for the use of enzymes whichleave 5′ or 3′ overhangs (e.g., BamHI, EcoRI, and HindIII) and thosewhich provide blunt ends (e.g., EcoRV, SnaBI, and StuI).

“Naturally occurring” refers to an endogenous polynucleotide orpolypeptide that may be isolated from viruses or prokaryotic oreukaryotic cells.

“Nucleic acid sequence” refers to the specific order of nucleotidesjoined by phosphodiester bonds in a linear, polymeric arrangement.Depending on the number of nucleotides, the nucleic acid sequence can beconsidered an oligomer, oligonucleotide, or polynucleotide. The nucleicacid can be DNA, RNA, or any nucleic acid analog, such as PNA, may be ofgenomic or synthetic origin, may be either double-stranded orsingle-stranded, and can represent either the sense or antisense(complementary) strand.

“Oligomer” refers to a nucleic acid sequence of at least about 6nucleotides and as many as about 60 nucleotides, preferably about 15 to40 nucleotides, and most preferably between about 20 and 30 nucleotides,that may be used in hybridization or amplification technologies.Oligomers may be used as, e.g., primers for PCR, and are usuallychemically synthesized.

“Operably linked” refers to the situation in which a first nucleic acidsequence is placed in a functional relationship with the second nucleicacid sequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences may be inclose proximity or contiguous and, where necessary to join two proteincoding regions, in the same reading frame.

“Peptide nucleic acid” (PNA) refers to a DNA mimic in which nucleotidebases are attached to a pseudopeptide backbone to increase stability.PNAs, also designated antigene agents, can prevent gene expression bytargeting complementary messenger RNA.

The phrases “percent identity” and “% identity”, as applied topolynucleotide sequences, refer to the percentage of residue matchesbetween at least two polynucleotide sequences aligned using astandardized algorithm Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences.

Percent identity between polynucleotide sequences may be determinedusing the default parameters of the CLUSTAL V algorithm as incorporatedinto the MEGALIGN version 3.12e sequence alignment program. This programis part of the LASERGENE software package, a suite of molecularbiological analysis programs (DNASTAR, Madison, Wis.). CLUSTAL V isdescribed in Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153 andin Higgins, D. G. et al. (1992) CABIOS 8:189-191. For pairwisealignments of polynucleotide sequences, the default parameters are setas follows: Ktuple=2, gap penalty=5, window=4, and “diagonals saved”=4.The “weighted” residue weight table is selected as the default. Percentidentity is reported by CLUSTAL V as the “percent similarity” betweenaligned polynucleotide sequence pairs.

Alternatively, a suite of commonly used and freely available sequencecomparison algorithms is provided by the National Center forBiotechnology Information (NCBI) Basic Local Alignment Search Tool(BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410), whichis available from several sources, including the NCBI, Bethesda, Md.,and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLASTsoftware suite includes various sequence analysis programs including“blastn,” that is used to determine alignment between a knownpolynucleotide sequence and other sequences on a variety of databases.Also available is a tool called “BLAST 2 Sequences” that is used fordirect pairwise comparison of two nucleotide sequences. “BLAST 2Sequences” can be accessed and used interactively athttp://www.ncbi.nlm.nih.gov/gorf/b12/. The “BLAST 2 Sequences” tool canbe used for both blastn and blastp (discussed below). BLAST programs arecommonly used with gap and other parameters set to default settings. Forexample, to compare two nucleotide sequences, one may use blastn withthe “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set at defaultparameters. Such default parameters may be, for example:

-   -   Matrix: BLOSUM62    -   Reward for match: 1    -   Penalty for mismatch: −2    -   Open Gap: 5 and Extension Gap: 2 penalties    -   Gap×drop-off: 50    -   Expect: 10    -   Word Size: 11    -   Filter: on

Percent identity may be measured over the length of an entire definedsequence, for example, as defined by a particular SEQ ID number, or maybe measured over a shorter length, for example, over the length of afragment taken from a larger, defined sequence, for instance, a fragmentof at least 20, at least 30, at least 40, at least 50, at least 70, atleast 100, or at least 200 contiguous nucleotides. Such lengths areexemplary only, and it is understood that any fragment length supportedby the sequences shown herein, in figures or Sequence Listings, may beused to describe a length over which percentage identity may bemeasured.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that all encode substantially the same protein.

The phrases “percent identity” and “% identity”, as applied topolypeptide sequences, refer to the percentage of residue matchesbetween at least two polypeptide sequences aligned using a standardizedalgorithm Methods of polypeptide sequence alignment are well-known. Somealignment methods take into account conservative amino acidsubstitutions. Such conservative substitutions, explained in more detailabove, generally preserve the hydrophobicity and acidity of thesubstituted residue, thus preserving the structure (and thereforefunction) of the folded polypeptide.

Percent identity between polypeptide sequences may be determined usingthe default parameters of the CLUSTAL V algorithm as incorporated intothe MEGALIGN version 3.12e sequence alignment program (described andreferenced above). For pairwise alignments of polypeptide sequencesusing CLUSTAL V, the default parameters are set as follows: Ktuple=1,gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix isselected as the default residue weight table. As with polynucleotidealignments, the percent identity is reported by CLUSTAL V as the“percent similarity” between aligned polypeptide sequence pairs.

Alternatively the NCBI BLAST software suite may be used. For example,for a pairwise comparison of two polypeptide sequences, one may use the“BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) with blastp set atdefault parameters. Such default parameters may be, for example:

-   -   Matrix: BLOSUM62    -   Open Gap: 11 and Extension Gap: 1 penalty    -   Gap×drop-off: 50    -   Expect: 10    -   Word Size: 3    -   Filter: on

Percent identity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in figures or SequenceListings, may be used to describe a length over which percentageidentity may be measured.

“Post-translational modification” of an MDDT may involve lipidation,glycosylation, phosphorylation, acetylation, racemization, proteolyticcleavage, and other modifications known in the art. These processes mayoccur synthetically or biochemically. Biochemical modifications willvary by cell type depending on the enzymatic milieu and the MDDT.

“Probe” refers to mddt or fragments thereof, which are used to detectidentical, allelic or related nucleic acid sequences. Probes areisolated oligonucleotides or polynucleotides attached to a detectablelabel or reporter molecule. Typical labels include radioactive isotopes,ligands, chemiluminescent agents, and enzymes. “Primers” are shortnucleic acids, usually DNA oligonucleotides, which may be annealed to atarget polynucleotide by complementary base-pairing. The primer may thenbe extended along the target DNA strand by a DNA polymerase enzyme.Primer pairs can be used for amplification (and identification) of anucleic acid sequence, e.g., by the polymerase chain reaction (PCR).

Probes and primers as used in the present invention typically compriseat least 15 contiguous nucleotides of a known sequence. In order toenhance specificity, longer probes and primers may also be employed,such as probes and primers that comprise at least 20, 30, 40, 50, 60,70, 80, 90, 100, or at least 150 consecutive nucleotides of thedisclosed nucleic acid sequences. Probes and primers may be considerablylonger than these examples, and it is understood that any lengthsupported by the specification, including the figures and SequenceListing, may be used.

Methods for preparing and using probes and primers are described in thereferences, for example Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press,Plainview, N.Y.; Ausubel et al., 1987, Current Protocols in MolecularBiology, Greene Publ. Assoc. & Wiley-Intersciences, New York, N.Y.;Innis et al., 1990, PCR Protocols, A Guide to Methods and Applications,Academic Press, San Diego, Calif. PCR primer pairs can be derived from aknown sequence, for example, by using computer programs intended forthat purpose such as Primer (Version 0.5, 1991, Whitehead Institute forBiomedical Research, Cambridge, Mass.).

Oligonucleotides for use as primers are selected using software known inthe art for such purpose. For example, OLIGO 4.06 software is useful forthe selection of PCR primer pairs of up to 100 nucleotides each, and forthe analysis of oligonucleotides and larger polynucleotides of up to5,000 nucleotides from an input polynucleotide sequence of up to 32kilobases. Similar primer selection programs have incorporatedadditional features for expanded capabilities. For example, the PrimOUprimer selection program (available to the public from the Genome Centerat University of Texas South West Medical Center, Dallas, Tex.) iscapable of choosing specific primers from megabase sequences and is thususeful for designing primers on a genome-wide scope. The Primer3 primerselection program (available to the public from the WhiteheadInstitute/MIT Center for Genome Research, Cambridge, Mass.) allows theuser to input a “mispriming library,” in which sequences to avoid asprimer binding sites are user-specified. Primer3 is useful, inparticular, for the selection of oligonucleotides for microarrays. (Thesource code for the latter two primer selection programs may also beobtained from their respective sources and modified to meet the user'sspecific needs.) The PrimeGen program (available to the public from theUK Human Genome Mapping Project Resource Centre, Cambridge UK) designsprimers based on multiple sequence alignments, thereby allowingselection of primers that hybridize to either the most conserved orleast conserved regions of aligned nucleic acid sequences. Hence, thisprogram is useful for identification of both unique and conservedoligonucleotides and polynucleotide fragments. The oligonucleotides andpolynucleotide fragments identified by any of the above selectionmethods are useful in hybridization technologies, for example, as PCR orsequencing primers, microarray elements, or specific probes to identifyfully or partially complementary polynucleotides in a sample of nucleicacids. Methods of oligonucleotide selection are not limited to thosedescribed above.

“Purified” refers to molecules, either polynucleotides or polypeptidesthat are isolated or separated from their natural environment and are atleast 60% free, preferably at least 75% free, and most preferably atleast 90% free from other compounds with which they are naturallyassociated.

A “recombinant nucleic acid” is a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo or more otherwise separated segments of sequence. This artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques such as those describedin Sambrook, supra. The term recombinant includes nucleic acids thathave been altered solely by addition, substitution, or deletion of aportion of the nucleic acid. Frequently, a recombinant nucleic acid mayinclude a nucleic acid sequence operably linked to a promoter sequence.Such a recombinant nucleic acid may be part of a vector that is used,for example, to transform a cell.

Alternatively, such recombinant nucleic acids may be part of a viralvector, e.g., based on a vaccinia virus, that could be use to vaccinatea mammal wherein the recombinant nucleic acid is expressed, inducing aprotective immunological response in the mammal.

“Regulatory element” refers to a nucleic acid sequence fromnontranslated regions of a gene, and includes enhancers, promoters,introns, and 3′ untranslated regions, which interact with host proteinsto carry out or regulate transcription or translation.

“Reporter” molecules are chemical or biochemical moieties used forlabeling a nucleic acid, an amino acid, or an antibody. They includeradionuclides; enzymes; fluorescent, chemiluminescent, or chromogenicagents; substrates; cofactors; inhibitors; magnetic particles; and othermoieties known in the art.

An “RNA equivalent,” in reference to a DNA sequence, is composed of thesame linear sequence of nucleotides as the reference DNA sequence withthe exception that all occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

“Sample” is used in its broadest sense. Samples may contain nucleic oramino acids, antibodies, or other materials, and may be derived from anysource (e.g., bodily fluids including, but not limited to, saliva,blood, and urine; chromosome(s), organelles, or membranes isolated froma cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate;and cleared cells or tissues or blots or imprints from such cells ortissues).

“Specific binding” or “specifically binding” refers to the interactionbetween a protein or peptide and its agonist, antibody, antagonist, orother binding partner. The interaction is dependent upon the presence ofa particular structure of the protein, e.g., the antigenic determinantor epitope, recognized by the binding molecule. For example, if anantibody is specific for epitope “A,” the presence of a polypeptidecontaining epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

“Substitution” refers to the replacement of at least one nucleotide oramino acid by a different nucleotide or amino acid.

“Substrate” refers to any suitable rigid or semi-rigid supportincluding, e.g., membranes, filters, chips, slides, wafers, fibers,magnetic or nonmagnetic beads, gels, tubing, plates, polymers,microparticles or capillaries. The substrate can have a variety ofsurface forms, such as wells, trenches, pins, channels and pores, towhich polynucleotides or polypeptides are bound.

A “transcript image” refers to the collective pattern of gene expressionby a particular tissue or cell type under given conditions at a giventime.

“Transformation” refers to a process by which exogenous DNA enters arecipient cell. Transformation may occur under natural or artificialconditions using various methods well known in the art. Transformationmay rely on any known method for the insertion of foreign nucleic acidsequences into a prokaryotic or eukaryotic host cell. The method isselected based on the host cell being transformed.

“Transformants” include stably transformed cells in which the insertedDNA is capable of replication either as an autonomously replicatingplasmid or as part of the host chromosome, as well as cells whichtransiently express inserted DNA or RNA.

A “transgenic organism,” as used herein, is any organism, including butnot limited to animals and plants, in which one or more of the cells ofthe organism contains heterologous nucleic acid introduced by way ofhuman intervention, such as by transgenic techniques well known in theart. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.The transgenic organisms contemplated in accordance with the presentinvention include bacteria, cyanobacteria, fungi, and plants andanimals. The isolated DNA of the present invention can be introducedinto the host by methods known in the art, for example infection,transfection, transformation or transconjugation. Techniques fortransferring the DNA of the present invention into such organisms arewidely known and provided in references such as Sambrook et al. (1989),supra.

A “variant” of a particular nucleic acid sequence is defined as anucleic acid sequence having at least 25% sequence identity to theparticular nucleic acid sequence over a certain length of one of thenucleic acid sequences using blastn with the “BLAST 2 Sequences” toolVersion 2.0.9 (May 7, 1999) set at default parameters. Such a pair ofnucleic acids may show, for example, at least 30%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95% oreven at least 98% or greater sequence identity over a certain definedlength. The variant may result in “conservative” amino acid changeswhich do not affect structural and/or chemical properties. A variant maybe described as, for example, an “allelic” (as defined above), “splice,”“species,” or “polymorphic” variant. A splice variant may havesignificant identity to a reference molecule, but will generally have agreater or lesser number of polynucleotides due to alternate splicing ofexons during mRNA processing. The corresponding polypeptide may possessadditional functional domains or lack domains that are present in thereference molecule. Species variants are polynucleotide sequences thatvary from one species to another. The resulting polypeptides generallywill have significant amino acid identity relative to each other. Apolymorphic variant is a variation in the polynucleotide sequence of aparticular gene between individuals of a given species. Polymorphicvariants also may encompass “single nucleotide polymorphisms” (SNPs) inwhich the polynucleotide sequence varies by one base. The presence ofSNPs may be indicative of, for example, a certain population, a diseasestate, or a propensity for a disease state.

In an alternative, variants of the polynucleotides of the presentinvention may be generated through recombinant methods. One possiblemethod is a DNA shuffling technique such as MOLECULARBREEDING (MaxygenInc., Santa Clara, Calif.; described in U.S. Pat. No. 5,837,458; Chang,C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F. C. etal. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996)Nat. Biotechnol. 14:315-319) to alter or improve the biologicalproperties of MDDT, such as its biological or enzymatic activity or itsability to bind to other molecules or compounds. DNA shuffling is aprocess by which a library of gene variants is produced usingPCR-mediated recombination of gene fragments. The library is thensubjected to selection or screening procedures that identify those genevariants with the desired properties. These preferred variants may thenbe pooled and further subjected to recursive rounds of DNA shuffling andselection/screening. Thus, genetic diversity is created through“artificial” breeding and rapid molecular evolution. For example,fragments of a single gene containing random point mutations may berecombined, screened, and then reshuffled until the desired propertiesare optimized. Alternatively, fragments of a given gene may berecombined with fragments of homologous genes in the same gene family,either from the same or different species, thereby maximizing thegenetic diversity of multiple naturally occurring genes in a directedand controllable manner.

A “variant” of a particular polypeptide sequence is defined as apolypeptide sequence having at least 40% sequence identity to theparticular polypeptide sequence over a certain length of one of thepolypeptide sequences using blastp with the “BLAST 2 Sequences” toolVersion 2.0.9 (May 7, 1999) set at default parameters. Such a pair ofpolypeptides may show, for example, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, or at least 98% orgreater sequence identity over a certain defined length of one of thepolypeptides.

THE INVENTION

In a particular embodiment, cDNA sequences derived from human tissuesand cell lines were aligned based on nucleotide sequence identity andassembled into “consensus” or “template” sequences which are designatedby the template identification numbers (template IDs) in column 2 ofTable 1. The sequence identification numbers (SEQ ID NO:s) correspondingto the template IDs are shown in column 1. The template sequences havesimilarity to GenBank sequences, or “hits,” as designated by the GINumbers in column 3. The statistical probability of each GenBank hit isindicated by a probability score in column 4, and the functionalannotation corresponding to each GenBank hit is listed in column 5.

The invention incorporates the nucleic acid sequences of these templatesas disclosed in the Sequence Listing and the use of these sequences inthe diagnosis and treatment of disease states characterized by defectsin disease detection and treatment molecules. The invention furtherutilizes these sequences in hybridization and amplificationtechnologies, and in particular, in technologies which assess geneexpression patterns correlated with specific cells or tissues and theirresponses in vivo or in vitro to pharmaceutical agents, toxins, andother treatments. In this manner, the sequences of the present inventionare used to develop a transcript image for a particular cell or tissue.

Derivation of Nucleic Acid Sequences

cDNA was isolated from libraries constructed using RNA derived fromnormal and diseased human tissues and cell lines. The human tissues andcell lines used for cDNA library construction were selected from a broadrange of sources to provide a diverse population of cDNAs representativeof gene transcription throughout the human body. Descriptions of thehuman tissues and cell lines used for cDNA library construction areprovided in the LIFESEQ database (Incyte Genomics, Inc. (Incyte), PaloAlto, Calif.). Human tissues were broadly selected from, for example,cardiovascular, dermatologic, endocrine, gastrointestinal,hematopoietic/immune system, musculoskeletal, neural, reproductive, andurologic sources.

Cell lines used for cDNA library construction were derived from, forexample, leukemic cells, teratocarcinomas, neuroepitheliomas, cervicalcarcinoma, lung fibroblasts, and endothelial cells. Such cell linesinclude, for example, THP-1, Jurkat, HUVEC, hNT2, WI38, HeLa, and othercell lines commonly used and available from public depositories(American Type Culture Collection, Manassas, Va.). Prior to mRNAisolation, cell lines were untreated, treated with a pharmaceuticalagent such as 5′-aza-2′-deoxycytidine, treated with an activating agentsuch as lipopolysaccharide in the case of leukocytic cell lines, or, inthe case of endothelial cell lines, subjected to shear stress.

Sequencing of the cDNAs

Methods for DNA sequencing are well known in the art. Conventionalenzymatic methods employ the Klenow fragment of DNA polymerase I,SEQUENASE DNA polymerase (U.S. Biochemical Corporation, Cleveland,Ohio), Taq polymerase (Applied Biosystems, Foster City, Calif.),thermostable T7 polymerase (Amersham Pharmacia Biotech, Inc. (AmershamPharmacia Biotech), Piscataway, N.J.), or combinations of polymerasesand proofreading exonucleases such as those found in the ELONGASEamplification system (Life Technologies Inc. (Life Technologies),Gaithersburg, Md.), to extend the nucleic acid sequence from anoligonucleotide primer annealed to the DNA template of interest. Methodshave been developed for the use of both single-stranded anddouble-stranded templates. Chain termination reaction products may beelectrophoresed on urea-polyacrylamide gels and detected either byautoradiography (for radioisotope-labeled nucleotides) or byfluorescence (for fluorophore-labeled nucleotides). Automated methodsfor mechanized reaction preparation, sequencing, and analysis usingfluorescence detection methods have been developed Machines used toprepare cDNAs for sequencing can include the MICROLAB 2200 liquidtransfer system (Hamilton Company (Hamilton), Reno, Nev.), Peltierthermal cycler (PTC200; MJ Research, Inc. (MJ Research), Watertown,Mass.), and ABI CATALYST 800 thermal cycler (Applied Biosystems).Sequencing can be carried out using, for example, the ABI 373 or 377(Applied Biosystems) or MEGABACE 1000 (Molecular Dynamics, Inc.(Molecular Dynamics), Sunnyvale, Calif.) DNA sequencing systems, orother automated and manual sequencing systems well known in the art.

The nucleotide sequences of the Sequence Listing have been prepared bycurrent, state-of-the-art, automated methods and, as such, may containoccasional sequencing errors or unidentified nucleotides. Suchunidentified nucleotides are designated by an N. These infrequentunidentified bases do not represent a hindrance to practicing theinvention for those skilled in the art. Several methods employingstandard recombinant techniques may be used to correct errors andcomplete the missing sequence information. (See, e.g., those describedin Ausubel, F. M. et al. (1997) Short Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y.; and Sambrook, J. et al. (1989)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y.)

Assembly of cDNA Sequences

Human polynucleotide sequences may be assembled using programs oralgorithms well known in the art. Sequences to be assembled are related,wholly or in part, and may be derived from a single or many differenttranscripts. Assembly of the sequences can be performed using suchprograms as PHRAP (Phils Revised Assembly Program) and the GELVIEWfragment assembly system (GCG), or other methods known in the art.

Alternatively, cDNA sequences are used as “component” sequences that areassembled into “template” or “consensus” sequences as follows. Sequencechromatograms are processed, verified, and quality scores are obtainedusing PHRED. Raw sequences are edited using an editing pathway known asBlock 1 (See, e.g., the LIFESEQ Assembled User Guide, Incyte Genomics,Palo Alto, Calif.). A series of BLAST comparisons is performed andlow-information segments and repetitive elements (e.g., dinucleotiderepeats, Alu repeats, etc.) are replaced by “n's”, or masked, to preventspurious matches. Mitochondrial and ribosomal RNA sequences are alsoremoved. The processed sequences are then loaded into a relationaldatabase management system (RDMS) which assigns edited sequences toexisting templates, if available. When additional sequences are addedinto the RDMS, a process is initiated which modifies existing templatesor creates new templates from works in progress (i.e., nonfinalassembled sequences) containing queued sequences or the sequencesthemselves. After the new sequences have been assigned to templates, thetemplates can be merged into bins. If multiple templates exist in onebin, the bin can be split and the templates reannotated.

Once gene bins have been generated based upon sequence alignments, binsare “clone joined” based upon clone information. Clone joining occurswhen the 5′ sequence of one clone is present in one bin and the 3′sequence from the same clone is present in a different bin, indicatingthat the two bins should be merged into a single bin. Only bins whichshare at least two different clones are merged.

A resultant template sequence may contain either a partial or a fulllength open reading frame, or all or part of a genetic regulatoryelement. This variation is due in part to the fact that the full lengthcDNAs of many genes are several hundred, and sometimes several thousand,bases in length. With current technology, cDNAs comprising the codingregions of large genes cannot be cloned because of vector limitations,incomplete reverse transcription of the mRNA, or incomplete “secondstrand” synthesis. Template sequences may be extended to includeadditional contiguous sequences derived from the parent RNA transcriptusing a variety of methods known to those of skill in the art. Extensionmay thus be used to achieve the full length coding sequence of a gene.

Analysis of the cDNA Sequences

The cDNA sequences are analyzed using a variety of programs andalgorithms which are well known in the art. (See, e.g., Ausubel, 1997,supra, Chapter 7.7; Meyers, R. A. (Ed.) (1995) Molecular Biology andBiotechnology, Wiley VCH, New York, N.Y., pp. 856-853; and Table 7.)These analyses comprise both reading frame determinations, e.g., basedon triplet codon periodicity for particular organisms (Fickett, J. W.(1982) Nucleic Acids Res. 10:5303-5318); analyses of potential start andstop codons; and homology searches.

Computer programs known to those of skill in the art for performingcomputer-assisted searches for amino acid and nucleic acid sequencesimilarity, include, for example, Basic Local Alignment Search Tool(BLAST; Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul, S. F.et al. (1990) J. Mol. Biol. 215:403-410). BLAST is especially useful indetermining exact matches and comparing two sequence fragments ofarbitrary but equal lengths, whose alignment is locally maximal and forwhich the alignment score meets or exceeds a threshold or cutoff scoreset by the user (Karlin, S. et al. (1988) Proc. Natl. Acad. Sci. USA85:841-845). Using an appropriate search tool (e.g., BLAST or HMM),GenBank, SwissProt, BLOCKS, PFAM and other databases may be searched forsequences containing regions of homology to a query mddt or MDDT of thepresent invention.

Other approaches to the identification, assembly, storage, and displayof nucleotide and polypeptide sequences are provided in “RelationalDatabase for Storing Biomolecule Information,” U.S. Ser. No. 08/947,845,filed Oct. 9, 1997; “Project-Based Full-Length Biomolecular SequenceDatabase,” U.S. Ser. No. 08/811,758, filed Mar. 6, 1997; and “RelationalDatabase and System for Storing Information Relating to BiomolecularSequences,” U.S. Ser. No. 09/034,807, filed Mar. 4, 1998, all of whichare incorporated by reference herein in their entirety.

Protein hierarchies can be assigned to the putative encoded polypeptidebased on, e.g., motif, BLAST, or biological analysis. Methods forassigning these hierarchies are described, for example, in “DatabaseSystem Employing Protein Function Hierarchies for Viewing BiomolecularSequence Data,” U.S. Ser. No. 08/812,290, filed Mar. 6, 1997,incorporated herein by reference.

Human Disease Detection and Treatment Molecule Sequences

The mddt of the present invention may be used for a variety ofdiagnostic and therapeutic purposes. For example, an mddt may be used todiagnose a particular condition, disease, or disorder associated withdisease detection and treatment molecules. Such conditions, diseases,and disorders include, but are not limited to, a cell proliferativedisorder, such as actinic keratosis, arteriosclerosis, atherosclerosis,bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD),myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,psoriasis, primary thrombocythemia, and cancers includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, a cancer of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; and anautoimmune/inflammatory disorder, such as actinic keratosis, acquiredimmunodeficiency syndrome (AIDS), Addison's disease, adult respiratorydistress syndrome, allergies, ankylosing spondylitis, amyloidosis,anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis,cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis,dermatomyositis, diabetes mellitus, emphysema, eryduroblastosis fetalis,erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture'ssyndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmalnocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable bowelsyndrome, episodic lymphopenia with lymphocytotoxins, mixed connectivetissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, myelofibrosis, osteoartritis, osteoporosis,pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter'ssyndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome,systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,primary thrombocythemia, thrombocytopenic purpura, ulcerative colitis,uveitis, Werner syndrome, complications of cancer, hemodialysis, andextracorporeal circulation, trauma, and hematopoietic cancer includinglymphoma, leukemia, and myeloma. The mddt can be used to detect thepresence of, or to quantify the amount of, an mddt-relatedpolynucleotide in a sample. This information is then compared toinformation obtained from appropriate reference samples, and a diagnosisis established. Alternatively, a polynucleotide complementary to a givenmddt can inhibit or inactivate a therapeutically relevant gene relatedto the mddt.

Analysis of mddt Expression Patterns

The expression of mddt may be routinely assessed by hybridization-basedmethods to determine, for example, the tissue-specificity,disease-specificity, or developmental stage-specificity of mddtexpression. For example, the level of expression of mddt may be comparedamong different cell types or tissues, among diseased and normal celltypes or tissues, among cell types or tissues at different developmentalstages, or among cell types or tissues undergoing various treatments.This type of analysis is useful, for example, to assess the relativelevels of mddt expression in fully or partially differentiated cells ortissues, to determine if changes in mddt expression levels arecorrelated with the development or progression of specific diseasestates, and to assess the response of a cell or tissue to a specifictherapy, for example, in pharmacological or toxicological studies.Methods for the analysis of mddt expression are based on hybridizationand amplification technologies and include membrane-based proceduressuch as northern blot analysis, high-throughput procedures that utilize,for example, microarrays, and PCR-based procedures.

Hybridization and Genetic Analysis

The mddt, their fragments, or complementary sequences, may be used toidentify the presence of and/or to determine the degree of similaritybetween two (or more) nucleic acid sequences. The mddt may be hybridizedto naturally occurring or recombinant nucleic acid sequences underappropriately selected temperatures and salt concentrations.Hybridization with a probe based on the nucleic acid sequence of atleast one of the mddt allows for the detection of nucleic acidsequences, including genomic sequences, which are identical or relatedto the mddt of the Sequence Listing. Probes may be selected fromnon-conserved or unique regions of at least one of the polynucleotidesof SEQ ID NO:1-45 and tested for their ability to identify or amplifythe target nucleic acid sequence using standard protocols.

Polynucleotide sequences that are capable of hybridizing, in particular,to those shown in SEQ ID NO:1-45 and fragments thereof, can beidentified using various conditions of stringency. (See, e.g., Wahl, G.M. and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R.(1987) Methods Enzymol. 152:507-511.) Hybridization conditions arediscussed in “Definitions.”

A probe for use in Souther or northern hybridization may be derived froma fragment of an mddt sequence, or its complement, that is up to severalhundred nucleotides in length and is either single-stranded ordouble-stranded. Such probes may be hybridized in solution to biologicalmaterials such as plasmids, bacterial, yeast, or human artificialchromosomes, cleared or sectioned tissues, or to artificial substratescontaining mddt. Microarrays are particularly suitable for identifyingthe presence of and detecting the level of expression for multiple genesof interest by examining gene expression correlated with, e.g., variousstages of development, treatment with a drug or compound, or diseaseprogression. An array analogous to a dot or slot blot may be used toarrange and link polynucleotides to the surface of a substrate using oneor more of the following: mechanical (vacuum), chemical, thermal, or UVbonding procedures. Such an array may contain any number of mddt and maybe produced by hand or by using available devices, materials, andmachines.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

Probes may be labeled by either PCR or enzymatic techniques using avariety of commercially available reporter molecules. For example,commercial kits are available for radioactive and chemiluminescentlabeling (Amersham Pharmacia Biotech) and for alkaline phosphataselabeling (Life Technologies). Alternatively, mddt may be cloned intocommercially available vectors for the production of RNA probes. Suchprobes may be transcribed in the presence of at least one labelednucleotide (e.g., ³²P-ATP, Amersham Pharmacia Biotech).

Additionally the polynucleotides of SEQ ID NO:1-45 or suitable fragmentsthereof can be used to isolate full length cDNA sequences utilizinghybridization and/or amplification procedures well known in the art,e.g., cDNA library screening, PCR amplification, etc. The molecularcloning of such full length cDNA sequences may employ the method of cDNAlibrary screening with probes using the hybridization, stringency,washing, and probing strategies described above and in Ausubel, supra,Chapters 3, 5, and 6. These procedures may also be employed with genomiclibraries to isolate genomic sequences of mddt in order to analyze,e.g., regulatory elements.

Genetic Mapping

Gene identification and mapping are important in the investigation andtreatment of almost all conditions, diseases, and disorders. Cancer,cardiovascular disease, Alzheimer's disease, arthritis, diabetes, andmental illnesses are of particular interest. Each of these conditions ismore complex than the single gene defects of sickle cell anemia orcystic fibrosis, with select groups of genes being predictive ofpredisposition for a particular condition, disease, or disorder. Forexample, cardiovascular disease may result from malfunctioning receptormolecules that fail to clear cholesterol from the bloodstream, anddiabetes may result when a particular individual's immune system isactivated by an infection and attacks the insulin-producing cells of thepancreas. In some studies, Alzheimer's disease has been linked to a geneon chromosome 21; other studies predict a different gene and location.Mapping of disease genes is a complex and reiterative process andgenerally proceeds from genetic linkage analysis to physical mapping.

As a condition is noted among members of a family, a genetic linkage maptraces parts of chromosomes that are inherited in the same pattern asthe condition. Statistics link the inheritance of particular conditionsto particular regions of chromosomes, as defined by RFLP or othermarkers. (See, for example, Lander, E. S. and Botstein, D. (1986) Proc.Natl. Acad. Sci. USA 83:7353-7357.) Occasionally, genetic markers andtheir locations are known from previous studies. More often, however,the markers are simply stretches of DNA that differ among individuals.Examples of genetic linkage maps can be found in various scientificjournals or at the Online Mendelian Inheritance in Man (OMIM) World WideWeb site.

In another embodiment of the invention, mddt sequences may be used togenerate hybridization probes useful in chromosomal mapping of naturallyoccurring genomic sequences. Either coding or noncoding sequences ofmddt may be used, and in some instances, noncoding sequences may bepreferable over coding sequences. For example, conservation of an mddtcoding sequence among members of a multi-gene family may potentiallycause undesired cross hybridization during chromosomal mapping. Thesequences may be mapped to a particular chromosome, to a specific regionof a chromosome, or to artificial chromosome constructions, e.g., humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions, orsingle chromosome cDNA libraries. (See, e.g., Harrington, J. J. et al.(1997) Nat. Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134;and Trask, B. J. (1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Meyers, supra, pp. 965-968.) Correlation between the location of mddt ona physical chromosomal map and a specific disorder, or a predispositionto a specific disorder, may help define the region of DNA associatedwith that disorder. The mddt sequences may also be used to detectpolymorphisms that are genetically liked to the inheritance of aparticular condition, disease, or disorder.

In situ hybridization of chromosomal preparations and genetic mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending existing genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of thecorresponding human chromosome is not known. These new marker sequencescan be mapped to human chromosomes and may provide valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once a disease or syndrome has beencrudely correlated by genetic linkage with a particular genomic region,e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to thatarea may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequences of the subject invention may alsobe used to detect differences in chromosomal architecture due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

Once a disease-associated gene is mapped to a chromosomal region, thegene must be cloned in order to identify mutations or other alterations(e.g., translocations or inversions) that may be correlated withdisease. This process requires a physical map of the chromosomal regioncontaining the disease-gene of interest along with associated markers. Aphysical map is necessary for determining the nucleotide sequence of andorder of marker genes on a particular chromosomal region. Physicalmapping techniques are well known in the art and require the generationof overlapping sets of cloned DNA fragments from a particular organelle,chromosome, or genome. These clones are analyzed to reconstruct andcatalog their order. Once the position of a marker is determined, theDNA from that region is obtained by consulting the catalog and selectingclones from that region. The gene of interest is located throughpositional cloning techniques using hybridization or similar methods.

Diagnostic Uses

The mddt of the present invention may be used to design probes useful indiagnostic assays. Such assays, well known to those skilled in the art,may be used to detect or confirm conditions, disorders, or diseasesassociated with abnormal levels of mddt expression. Labeled probesdeveloped from mddt sequences are added to a sample under hybridizingconditions of desired stringency. In some instances, mddt, or fragmentsor oligonucleotides derived from mddt, may be used as primers inamplification steps prior to hybridization. The amount of hybridizationcomplex formed is quantified and compared with standards for that cellor tissue. If mddt expression varies significantly from the standard,the assay indicates the presence of the condition, disorder, or disease.Qualitative or quantitative diagnostic methods may include northern, dotblot, or other membrane or dip-stick based technologies ormultiple-sample format technologies such as PCR, enzyme-linkedimmunosorbent assay (ELISA)-like, pin, or chip-based assays.

The probes described above may also be used to monitor the progress ofconditions, disorders, or diseases associated with abnormal levels ofmddt expression, or to evaluate the efficacy of a particular therapeutictreatment The candidate probe may be identified from the mddt that arespecific to a given human tissue and have not been observed in GenBankor other genome databases. Such a probe may be used in animal studies,preclinical tests, clinical trials, or in monitoring the treatment of anindividual patient In a typical process, standard expression isestablished by methods well known in the art for use as a basis ofcomparison, samples from patients affected by the disorder or diseaseare combined with the probe to evaluate any deviation from the standardprofile, and a therapeutic agent is administered and effects aremonitored to generate a treatment profile. Efficacy is evaluated bydetermining whether the expression progresses toward or returns to thestandard normal pattern. Treatment profiles may be generated over aperiod of several days or several months. Statistical methods well knownto those skilled in the art may be use to determine the significance ofsuch therapeutic agents.

The polynucleotides are also useful for identifying individuals fromminute biological samples, for example, by matching the RFLP pattern ofa sample's DNA to that of an individual's DNA. The polynucleotides ofthe present invention can also be used to determine the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. These sequences can be used to prepare PCR primers foramplifying and isolating such selected DNA, which can then be sequenced.Using this technique, an individual can be identified through a uniqueset of DNA sequences. Once a unique ID database is established for anindividual, positive identification of that individual can be made fromextremely small tissue samples.

In a particular aspect, oligonucleotide primers derived from the mddt ofthe invention may be used to detect single nucleotide polymorphisms(SNPs). SNPs are substitutions, insertions and deletions that are afrequent cause of inherited or acquired genetic disease in humans.Methods of SNP detection include, but are not limited to,single-stranded conformation polymorphism (SSCP) and fluorescent SSCP(fSSCP) methods. In SSCP, oligonucleotide primers derived from mddt areused to amplify DNA using the polymerase chain reaction (PCR). The DNAmay be derived, for example, from diseased or normal tissue, biopsysamples, bodily fluids, and the like. SNPs in the DNA cause differencesin the secondary and tertiary structures of PCR products insingle-stranded form, and these differences are detectable using gelelectrophoresis in non-denaturing gels. In fSCCP, the oligonucleotideprimers are fluorescently labeled, which allows detection of theamplimers in high-throughput equipment such as DNA sequencing machines.Additionally, sequence database analysis methods, termed in silico SNP(isSNP), are capable of identifying polymorphisms by comparing thesequences of individual overlapping DNA fragments which assemble into acommon consensus sequence. These computer-based methods filter outsequence variations due to laboratory preparation of DNA and sequencingerrors using statistical models and automated analyses of DNA sequencechromatograms. In the alternative, SNPs may be detected andcharacterized by mass spectrometry using, for example, the highthroughput MASSARRAY system (Sequenom, Inc., San Diego, Calif.).

DNA-based identification techniques are critical in forensic technology.DNA sequences taken from very small biological samples such as tissues,e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, etc.,can be amplified using, e.g., PCR, to identify individuals. (See, e.g.,Erlich, H. (1992) PCR Technology, Freeman and Co., New York, N.Y.).Similarly, polynucleotides of the present invention can be used aspolymorphic markers.

There is also a need for reagents capable of identifying the source of aparticular tissue. Appropriate reagents can comprise, for example, DNAprobes or primers prepared from the sequences of the present inventionthat are specific for particular tissues. Panels of such reagents canidentify tissue by species and/or by organ type. In a similar fashion,these reagents can be used to screen tissue cultures for contamination.

The polynucleotides of the present invention can also be used asmolecular weight markers on nucleic acid gels or Southern blots, asdiagnostic probes for the presence of a specific mRNA in a particularcell type, in the creation of subtracted cDNA libraries which aid in thediscovery of novel polynucleotides, in selection and synthesis ofoligomers for attachment to an array or other support, and as an antigento elicit an immune response.

Disease Model Systems Using mddt

The mddt of the invention or their mammalian homologs may be “knockedout” in an animal model system using homologous recombination inembryonic stem (ES) cells. Such techniques are well known in the art andare useful for the generation of animal models of human disease. (See,e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No. 5,767,337.) For example,mouse ES cells, such as the mouse 129/SvJ cell line, are derived fromthe early mouse embryo and grown in culture. The ES cells aretransformed with a vector containing the gene of interest disrupted by amarker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi,M. R. (1989) Science 244:1288-1292). The vector integrates into thecorresponding region of the host genome by homologous recombination.Alternatively, homologous recombination takes place using the Cre-loxPsystem to knockout a gene of interest in a tissue- or developmentalstage-specific manner (Marth, J. D. (1996) Clin. Invest. 97:1999-2002;Wagner, K. U. et al. (1997) Nucleic Acids Res. 25:4323-4330).Transformed ES cells are identified and microinjected into mouse cellblastocysts such as those from the C57BL/6 mouse strain. The blastocystsare surgically transferred to pseudopregnant dams, and the resultingchimeric progeny are genotyped and bred to produce heterozygous orhomozygous strains. Transgenic animals thus generated may be tested withpotential therapeutic or toxic agents.

The mddt of the invention may also be manipulated in vitro in ES cellsderived from human blastocysts. Human ES cells have the potential todifferentiate into at least eight separate cell lineages includingendoderm, mesoderm, and ectodermal cell types. These cell lineagesdifferentiate into, for example, neural cells, hematopoietic lineages,and cardiomyocytes (Thomson, J. A. et al. (1998) Science 282:1145-1147).

The mddt of the invention can also be used to create “knockin” humanizedanimals (pigs) or transgenic animals (mice or rats) to model humandisease. With knockin technology, a region of mddt is injected intoanimal ES cells, and the injected sequence integrates into the animalcell genome. Transformed cells are injected into blastulae, and theblastulae are implanted as described above. Transgenic progeny or inbredlines are studied and treated with potential pharmaceutical agents toobtain information on treatment of a human disease. Alternatively, amammal inbred to overexpress mddt, resulting, e.g., in the secretion ofMDDT in its milk, may also serve as a convenient source of that protein(Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).

Screening Assays

MDDT encoded by polynucleotides of the present invention may be used toscreen for molecules that bind to or are bound by the encodedpolypeptides. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the bound molecule. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors), orsmall molecules.

Preferably, the molecule is closely related to the natural ligand of thepolypeptide, e.g., a ligand or fragment thereof, a natural substrate, ora structural or functional mimetic. (See, Coligan et al., (1991) CurrentProtocols in Immunology 1(2): Chapter 5.) Similarly, the molecule can beclosely related to the natural receptor to which the polypeptide binds,or to at least a fragment of the receptor, e.g., the active site. Ineither case, the molecule can be rationally designed using knowntechniques. Preferably, the screening for these molecules involvesproducing appropriate cells which express the polypeptide, either as asecreted protein or on the cell membrane. Preferred cells include cellsfrom mammals, yeast, Drosophila, or E. coli. Cells expressing thepolypeptide or cell membrane fractions which contain the expressedpolypeptide are then contacted with a test compound and binding,stimulation, or inhibition of activity of either the polypeptide or themolecule is analyzed.

An assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a fluorophore, radioisotope,enzyme conjugate, or other detectable label. Alternatively, the assaymay assess binding in the presence of a labeled competitor.

Additionally, the assay can be carried out using cell-free preparations,polypeptide/molecule affixed to a solid support, chemical libraries, ornatural product mixtures. The assay may also simply comprise the stepsof mixing a candidate compound with a solution containing a polypeptide,measuring polypeptide/molecule activity or binding, and comparing thepolypeptide/molecule activity or binding to a standard.

Preferably, an ELISA assay using, e.g., a monoclonal or polyclonalantibody, can measure polypeptide level in a sample. The antibody canmeasure polypeptide level by either binding, directly or indirectly, tothe polypeptide or by competing with the polypeptide for a substrate.

All of the above assays can be used in a diagnostic or prognosticcontext. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting thepolypeptide/molecule. Moreover, the assays can discover agents which mayinhibit or enhance the production of the polypeptide from suitablymanipulated cells or tissues.

Transcript Imaging and Toxicological Testing

Another embodiment relates to the use of mddt to develop a transcriptimage of a tissue or cell type. A transcript image represents the globalpattern of gene expression by a particular tissue or cell type. Globalgene expression patterns are analyzed by quantifying the number ofexpressed genes and their relative abundance under given conditions andat a given time. (See Seilhamer et al., “Comparative Gene TranscriptAnalysis,” U.S. Pat. No. 5,840,484, expressly incorporated by referenceherein.) Thus a transcript image may be generated by hybridizing thepolynucleotides of the present invention or their complements to thetotality of transcripts or reverse transcripts of a particular tissue orcell type. In one embodiment, the hybridization takes place inhigh-throughput format, wherein the polynucleotides of the presentinvention or their complements comprise a subset of a plurality ofelements on a microarray. The resultant transcript image would provide aprofile of gene activity pertaining to disease detection and treatmentmolecules.

Transcript images which profile mddt expression may be generated usingtranscripts isolated from tissues, cell lines, biopsies, or otherbiological samples. The transcript image may thus reflect mddtexpression in vivo, as in the case of a tissue or biopsy sample, or invitro, as in the case of a cell line.

Transcript images which profile mddt expression may also be used inconjunction with in vitro model systems and preclinical evaluation ofpharmaceuticals, as well as toxicological testing of industrial andnaturally-occurring environmental compounds. All compounds inducecharacteristic gene expression patterns, frequently termed molecularfingerprints or toxicant signatures, which are indicative of mechanismsof action and toxicity (Nuwaysir, E. F. et al. (1999) Mol. Carcinog.24:153-159; Steiner, S. and Anderson, N. L. (2000) Toxicol. Lett.112-113:467-71, expressly incorporated by reference herein). If a testcompound has a signature similar to that of a compound with knowntoxicity, it is likely to share those toxic properties. Thesefingerprints or signatures are most useful and refined when they containexpression information from a large number of genes and gene families.Ideally, a genome-wide measurement of expression provides the highestquality signature. Even genes whose expression is not altered by anytested compounds are important as well, as the levels of expression ofthese genes are used to normalize the rest of the expression data. Thenormalization procedure is useful for comparison of expression dataafter treatment with different compounds. While the assignment of genefunction to elements of a toxicant signature aids in interpretation oftoxicity mechanisms, knowledge of gene function is not necessary for thestatistical matching of signatures which leads to prediction oftoxicity. (See, for example, Press Release 00-02 from the NationalInstitute of Environmental Health Sciences, released Feb. 29, 2000,available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore,it is important and desirable in toxicological screening using toxicantsignatures to include all expressed gene sequences.

In one embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing nucleic acids with the testcompound Nucleic acids that are expressed in the treated biologicalsample are hybridized with one or more probes specific to thepolynucleotides of the present invention, so that transcript levelscorresponding to the polynucleotides of the present invention may bequantified. The transcript levels in the treated biological sample arecompared with levels in an untreated biological sample. Differences inthe transcript levels between the two samples are indicative of a toxicresponse caused by the test compound in the treated sample.

Another particular embodiment relates to the use of MDDT encoded bypolynucleotides of the present invention to analyze the proteome of atissue or cell type. The term proteome refers to the global pattern ofprotein expression in a particular tissue or cell type. Each proteincomponent of a proteome can be subjected individually to furtheranalysis. Proteome expression patterns, or profiles, are analyzed byquantifying the number of expressed proteins and their relativeabundance under given conditions and at a given time. A profile of acell's proteome may thus be generated by separating and analyzing thepolypeptides of a particular tissue or cell type. In one embodiment, theseparation is achieved using two-dimensional gel electrophoresis, inwhich proteins from a sample are separated by isoelectric focusing inthe first dimension, and then according to molecular weight by sodiumdodecyl sulfate slab gel electrophoresis in the second dimension(Steiner and Anderson, supra). The proteins are visualized in the gel asdiscrete and uniquely positioned spots, typically by staining the gelwith an agent such as Coomassie Blue or silver or fluorescent stains.The optical density of each protein spot is generally proportional tothe level of the protein in the sample. The optical densities ofequivalently positioned protein spots from different samples, forexample, from biological samples either treated or untreated with a testcompound or therapeutic agent, are compared to identify any changes inprotein spot density related to the treatment. The proteins in the spotsare partially sequenced using, for example, standard methods employingchemical or enzymatic cleavage followed by mass spectrometry. Theidentity of the protein in a spot may be determined by comparing itspartial sequence, preferably of at least 5 contiguous amino acidresidues, to the polypeptide sequences of the present invention. In somecases, further sequence data may be obtained for definitive proteinidentification.

A proteomic profile may also be generated using antibodies specific forMDDT to quantify the levels of MDDT expression. In one embodiment, theantibodies are used as elements on a microarray, and protein expressionlevels are quantified by exposing the microarray to the sample anddetecting the levels of protein bound to each array element (Lueking, A.et al. (1999) Anal. Biochem. 270:103-11; Mendoze, L. G. et al. (1999)Biotechniques 27:778-88). Detection may be performed by a variety ofmethods known in the art, for example, by reacting the proteins in thesample with a thiol- or amino-reactive fluorescent compound anddetecting the amount of fluorescence bound at each array element.

Toxicant signatures at the proteome level are also useful fortoxicological screening, and should be analyzed in parallel withtoxicant signatures at the transcript level. There is a poor correlationbetween transcript and protein abundances for some proteins in sometissues (Anderson, N. L. and Seilhamer, J. (1997) Electrophoresis18:533-537), so proteome toxicant signatures may be useful in theanalysis of compounds which do not significantly affect the transcriptimage, but which alter the proteomic profile. In addition, the analysisof transcripts in body fluids is difficult, due to rapid degradation ofmRNA, so proteomic profiling may be more reliable and informative insuch cases.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compoundProteins that are expressed in the treated biological sample areseparated so that the amount of each protein can be quantified. Theamount of each protein is compared to the amount of the correspondingprotein in an untreated biological sample. A difference in the amount ofprotein between the two samples is indicative of a toxic response to thetest compound in the treated sample. Individual proteins are identifiedby sequencing the amino acid residues of the individual proteins andcomparing these partial sequences to the MDDT encoded by polynucleotidesof the present invention.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins from the biological sample are incubated with antibodiesspecific to the MDDT encoded by polynucleotides of the presentinvention. The amount of protein recognized by the antibodies isquantified. The amount of protein in the treated biological sample iscompared with the amount in an untreated biological sample. A differencein the amount of protein between the two samples is indicative of atoxic response to the test compound in the treated sample.

Transcript images may be used to profile mddt expression in distincttissue types. This process can be used to determine disease detectionand treatment molecule activity in a particular tissue type relative tothis activity in a different tissue type. Transcript images may be usedto generate a profile of mddt expression characteristic of diseasedtissue. Transcript images of tissues before and after treatment may beused for diagnostic purposes, to monitor the progression of disease, andto monitor the efficacy of drug treatments for diseases which affect theactivity of disease detection and treatment molecules.

Transcript images of cell lines can be used to assess disease detectionand treatment molecule activity and/or to identify cell lines that lackor misregulate this activity. Such cell lines may then be treated withpharmaceutical agents, and a transcript image following treatment mayindicate the efficacy of these agents in restoring desired levels ofthis activity. A similar approach may be used to assess the toxicity ofpharmaceutical agents as reflected by undesirable changes in diseasedetection and treatment molecule activity. Candidate pharmaceuticalagents may be evaluated by comparing their associated transcript imageswith those of pharmaceutical agents of known effectiveness.

Antisense Molecules

The polynucleotides of the present invention are useful in antisensetechnology. Antisense technology or therapy relies on the modulation ofexpression of a target protein through the specific binding of anantisense sequence to a target sequence encoding the target protein ordirecting its expression. (See, e.g., Agrawal, S., ed. (1996) AntisenseTherapeutics, Humana Press Inc., Totawa, N.J.; Alama, A. et al. (1997)Pharmacol. Res. 36(3):171-178; Crooke, S. T. (1997) Adv. Pharmacol.40:1-49; Sharma, H. W. and R. Narayanan (1995) Bioessays17(12):1055-1063; and Lavrosky, Y. et al. (1997) Biochem. Mol. Med.62(1):11-22.) An antisense sequence is a polynucleotide sequence capableof specifically hybridizing to at least a portion of the targetsequence. Antisense sequences bind to cellular mRNA and/or genomic DNA,affecting translation and/or transcription. Antisense sequences can beDNA, RNA, or nucleic acid mimics and analogs. (See, e.g., Rossi, J. J.et al. (1991) Antisense Res. Dev. 1(3):285-288; Lee, R. et al. (1998)Biochemistry 37(3):900-1010; Pardridge, W. M. et al. (1995) Proc. Natl.Acad. Sci. USA 92(12):5592-5596; and Nielsen, P. E. and Haaima, G.(1997) Chem. Soc. Rev. 96:73-78.) Typically, the binding which resultsin modulation of expression occurs through hybridization or binding ofcomplementary base pairs. Antisense sequences can also bind to DNAduplexes through specific interactions in the major groove of the doublehelix.

The polynucleotides of the present invention and fragments thereof canbe used as antisense sequences to modify the expression of thepolypeptide encoded by mddt The antisense sequences can be produced exvivo, such as by using any of the ABI nucleic acid synthesizer series(Applied Biosystems) or other automated systems known in the art.Antisense sequences can also be produced biologically, such as bytransforming an appropriate host cell with an expression vectorcontaining the sequence of interest. (See, e.g., Agrawal, supra.)

In therapeutic use, any gene delivery system suitable for introductionof the antisense sequences into appropriate target cells can be used.Antisense sequences can be delivered intracellularly in the form of anexpression plasmid which, upon transcription, produces a sequencecomplementary to at least a portion of the cellular sequence encodingthe target protein. (See, e.g., Slater, J. E., et al. (1998) J. AllergyClin. Immunol. 102(3):469-475; and Scanlon, K. J., et al. (1995)9(13):1288-1296.) Antisense sequences can also be introducedintracellularly through the use of viral vectors, such as retrovirus andadeno-associated virus vectors. (See, e.g., Miller, A. D. (1990) Blood76:271; Ausubel, F. M. et al. (1995) Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y.; Uckert, W. and W. Walther(1994) Pharmacol. Ther. 63(3):323-347.) Other gene delivery mechanismsinclude liposome-derived systems, artificial viral envelopes, and othersystems known in the art. (See, e.g., Rossi, J. J. (1995) Br. Med. Bull.51(1):217-225; Boado, R. J. et al. (1998) J. Pharm. Sci.87(11):1308-1315; and Morris, M. C. et al. (1997) Nucleic Acids Res.25(14):2730-2736.)

Expression

In order to express a biologically active MDDT, the nucleotide sequencesencoding MDDT or fragments thereof may be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor transcriptional and translational control of the inserted codingsequence in a suitable host. Methods which are well known to thoseskilled in the art may be used to construct expression vectorscontaining sequences encoding MDDT and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. (See, e.g., Sambrook, supra, Chapters 4, 8, 16, and 17;and Ausubel, supra, Chapters 9, 10, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding MDDT. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal (mammalian) cellsystems. (See, e.g., Sambrook, supra; Ausubel, 1995, supra, Van Heeke,G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Bitter, G. A.et al. (1987) Methods Enzymol. 153:516-544; Scorer, C. A. et al. (1994)Bio/Technology 12:181-184; Engelhard, E. K. et al. (1994) Proc. Natl.Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther.7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; Coruzzi, G. et al.(1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ.17:85-105; The McGraw Hill Yearbook of Science and Technology (1992)McGraw Hill, New York, N.Y., pp. 191-196; Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. USA 81:3655-3659; and Harrington, J. J. et al.(1997) Nat. Genet. 15:345-355.) Expression vectors derived fromretroviruses, adenoviruses, or herpes or vaccinia viruses, or fromvarious bacterial plasmids, may be used for delivery of nucleotidesequences to the targeted organ, tissue, or cell population. (See, e.g.,Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. etal., (1993) Proc. Natl. Acad. Sci. USA 90(13):6340-6344; Buller, R. M.et al. (1985) Nature 317(6040):813-815; McGregor, D. P. et al. (1994)Mol. Immunol. 31(3):219-226; and Verma, I. M. and N. Somia (1997) Nature389:239-242.) The invention is not limited by the host cell employed.

For long term production of recombinant proteins in mammalian systems,stable expression of MDDT in cell lines is preferred. For example,sequences encoding MDDT can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Any number of selection systems may be used torecover transformed cell lines. (See, e.g., Wigler, M. et al. (1977)Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.; Wigler, M. etal. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F.et al. (1981) J. Mol. Biol. 150:1-14; Hartman, S. C. and R. C. Mulligan(1988) Proc. Natl. Acad. Sci. USA 85:8047-8051; Rhodes, C. A. (1995)Methods Mol. Biol. 55:121-131.)

Therapeutic Uses of mddt

The mddt of the invention may be used for somatic or germline genetherapy. Gene therapy may be performed to (i) correct a geneticdeficiency (e.g., in the cases of severe combined immunodeficiency(SCID)-X1 disease characterized by X-linked inheritance(Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combinedimmunodeficiency syndrome associated with an inherited adenosinedeaminase (ADA) deficiency (Blaese, R. M. et al. (1995) Science270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cysticfibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R. G. etal. (1995) Hum. Gene Therapy 6:643-666; Crystal, R. G. et al. (1995)Hum. Gene Therapy 6:667-703), thalassemias, familialhypercholesterolemia, and hemophilia resulting from Factor VIII orFactor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410;Verma, I. M. and Somia, N. (1997) Nature 389:239-242)), (ii) express aconditionally lethal gene product (e.g., in the case of cancers whichresult from unregulated cell proliferation), or (iii) express a proteinwhich affords protection against intracellular parasites (e.g., againsthuman retroviruses, such as human immunodeficiency virus (HIV)(Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996)Proc. Natl. Acad. Sci. USA. 93:11395-11399), hepatitis B or C virus(HBV, HCV); fungal parasites, such as Candida albicans andParacoccidioides brasiliensis; and protozoan parasites such asPlasmodium falcidarum and Trypanosoma cruzi). In the case where agenetic deficiency in mddt expression or regulation causes disease, theexpression of mddt from an appropriate population of transduced cellsmay alleviate the clinical manifestations caused by the geneticdeficiency.

In a further embodiment of the invention, diseases or disorders causedby deficiencies in mddt are treated by constructing mammalian expressionvectors comprising mddt and introducing these vectors by mechanicalmeans into mddt-deficient cells. Mechanical transfer technologies foruse with cells in vivo or ex vitro include (i) direct DNA microinjectioninto individual cells, (ii) ballistic gold particle delivery, (iii)liposome-mediated transfection, (iv) receptor-mediated gene transfer,and (v) the use of DNA transposons (Morgan, R. A. and Anderson, W. F.(1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510;Boulay, J-L. and Récipon, H. (1998) Curr. Opin. Biotechnol. 9:445-450).

Expression vectors that may be effective for the expression of mddtinclude, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP,PVAX vectors (Invitrogen, Carlsbad, Calif.), PCMV-SCRIPT, PCMV-TAG,PEGSH/PERV (Stratagene, La Jolla, Calif.), and PTET-OFF, PTET-ON, PTRE2,PTRE2-LUC, PTK-HYG (Clontech, Palo Alto, Calif.). The mddt of theinvention may be expressed using (i) a constitutively active promoter,(e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus,thymidine kinase (TK), or β-actin genes), (ii) an inducible promoter(e.g., the tetracycline-regulated promoter (Gossen, M. and Bujard, H.(1992) Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551; Gossen, M. et al.,(1995) Science 268:1766-1769; Rossi, F. M. V. and Blau, H. M. (1998)Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REXplasmid (Invitrogen); the ecdysone-inducible promoter (available in theplasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin induciblepromoter; or the RU486/mifepristone inducible promoter (Rossi, F. M. V.and Blau, H. M. supra), or (iii) a tissue-specific promoter or thenative promoter of the endogenous gene encoding MDDT from a normalindividual.

Commercially available liposome transformation kits (e.g., the PERFECTLIPID TRANSFECTION KIT, available from Invitrogen) allow one withordinary skill in the art to deliver polynucleotides to target cells inculture and require minimal effort to optimize experimental parameters.In the alternative, transformation is performed using the calciumphosphate method (Graham, F. L. and Eb, A. J. (1973) Virology52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J.1:841-845). The introduction of DNA to primary cells requiresmodification of these standardized mammalian transfection protocols.

In another embodiment of the invention, diseases or disorders caused bygenetic defects with respect to mddt expression are treated byconstructing a retrovirus vector consisting of (i) mddt under thecontrol of an independent promoter or the retrovirus long terminalrepeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii)a Rev-responsive element (RRE) along with additional retroviruscis-acting RNA sequences and coding sequences required for efficientvector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) arecommercially available (Stratagene) and are based on published data(Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92:6733-6737),incorporated by reference herein. The vector is propagated in anappropriate vector producing cell line (VPCL) that expresses an envelopegene with a tropism for receptors on the target cells or a promiscuousenvelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol.61:1647-1650; Bender, M. A. et al. (1987) J. Virol. 61:1639-1646; Adam,M. A. and Miller, A. D. (1988) J. Virol. 62:3802-3806; Dull, T. et al.(1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J. Virol.72:9873-9880). U.S. Pat. No. 5,910,434 to Rigg (“Method for obtainingretrovirus packaging cell lines producing high transducing efficiencyretroviral supernatant”) discloses a method for obtaining retroviruspackaging cell lines and is hereby incorporated by reference.Propagation of retrovirus vectors, transduction of a population of cells(e.g., CD4⁺ T-cells), and the return of transduced cells to a patientare procedures well known to persons skilled in the art of gene therapyand have been well documented (Ranga, U. et al. (1997) J. Virol.71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716; Ranga, U. et al. (1998) Proc. Natl.Acad. Sci. U.S.A. 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).

In the alternative, an adenovirus-based gene therapy delivery system isused to deliver mddt to cells which have one or more geneticabnormalities with respect to the expression of mddt. The constructionand packaging of adenovirus-based vectors are well known to those withordinary skill in the art. Replication defective adenovirus vectors haveproven to be versatile for importing genes encoding immunoregulatoryproteins into intact islets in the pancreas (Csete, M. E. et al. (1995)Transplantation 27:263-268). Potentially useful adenoviral vectors aredescribed in U.S. Pat. No. 5,707,618 to Armentano (“Adenovirus vectorsfor gene therapy”), hereby incorporated by reference. For adenoviralvectors, see also Antinozzi, P. A. et al. (1999) Annu. Rev. Nutr.19:511-544 and Verma, I. M. and Somia, N. (1997) Nature 18:389:239-242,both incorporated by reference herein.

In another alternative, a herpes-based, gene therapy delivery system isused to deliver mddt to target cells which have one or more geneticabnormalities with respect to the expression of mddt. The use of herpessimplex virus (HSV)-based vectors may be especially valuable forintroducing mddt to cells of the central nervous system, for which HSVhas a tropism. The construction and packaging of herpes-based vectorsare well known to those with ordinary skill in the art. Areplication-competent herpes simplex virus (HSV) type 1-based vector hasbeen used to deliver a reporter gene to the eyes of primates (Liu, X. etal. (1999) Exp. Eye Res.169:385-395). The construction of a HSV-1 virusvector has also been disclosed in detail in U.S. Pat. No. 5,804,413 toDeLuca (“Herpes simplex virus strains for gene transfer”), which ishereby incorporated by reference. U.S. Pat. No. 5,804,413 teaches theuse of recombinant HSV d92 which consists of a genome containing atleast one exogenous gene to be transferred to a cell under the controlof the appropriate promoter for purposes including human gene therapy.Also taught by this patent are the construction and use of recombinantHSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see alsoGoins, W. F. et al. 1999 J. Virol. 73:519-532 and Xu, H. et al., (1994)Dev. Biol. 163:152-161, hereby incorporated by reference. Themanipulation of cloned herpesvirus sequences, the generation ofrecombinant virus following the transfection of multiple plasmidscontaining different segments of the large herpesvirus genomes, thegrowth and propagation of herpesvirus, and the infection of cells withherpesvirus are techniques well known to those of ordinary skill in theart.

In another alternative, an alphavirus (positive, single-stranded RNAvirus) vector is used to deliver mddt to target cells. The biology ofthe prototypic alphavirus, Semliki Forest Virus (SFV), has been studiedextensively and gene transfer vectors have been based on the SFV genome(Garoff, H. and Li, K-J. (1998) Curr. Opin. Biotech. 9:464-469). Duringalphavirus RNA replication, a subgenomic RNA is generated that normallyencodes the viral capsid proteins. This subgenomic RNA replicates tohigher levels than the full-length genomic RNA, resulting in theoverproduction of capsid proteins relative to the viral proteins withenzymatic activity (e.g., protease and polymerase). Similarly, insertingmddt into the alphavirus genome in place of the capsid-coding regionresults in the production of a large number of mddt RNAs and thesynthesis of high levels of MDDT in vector transduced cells. Whilealphavirus infection is typically associated with cell lysis within afew days, the ability to establish a persistent infection in hamsternormal kidney cells (BHK-21) with a variant of Sindbis virus (SIN)indicates that the lytic replication of alphaviruses can be altered tosuit the needs of the gene therapy application (Dryga, S. A. et al.(1997) Virology 228:74-83). The wide host range of alphaviruses willallow the introduction of mddt into a variety of cell types. Thespecific transduction of a subset of cells in a population may requirethe sorting of cells prior to transduction. The methods of manipulatinginfectious cDNA clones of alphaviruses, performing alphavirus cDNA andRNA transfections, and performing alphavirus infections, are well knownto those with ordinary skill in the art.

Antibodies

Anti-MDDT antibodies may be used to analyze protein expression levels.Such antibodies include, but are not limited to, polyclonal, monoclonal,chimeric, single chain, and Fab fragments. For descriptions of andprotocols of antibody technologies, see, e.g., Pound J. D. (1998)Immunochemical Protocols, Humana Press, Totowa, N.J.

The amino acid sequence encoded by the mddt of the Sequence Listing maybe analyzed by appropriate software (e.g., LASERGENE NAVIGATOR software,DNASTAR) to determine regions of high immunogenicity. The optimalsequences for immunization are selected from the C-terminus, theN-terminus, and those intervening, hydrophilic regions of thepolypeptide which are likely to be exposed to the external environmentwhen the polypeptide is in its natural conformation. Analysis used toselect appropriate epitopes is also described by Ausubel (1997, supra,Chapter 11.7). Peptides used for antibody induction do not need to havebiological activity; however, they must be antigenic. Peptides used toinduce specific antibodies may have an amino acid sequence consisting ofat least five amino acids, preferably at least 10 amino acids, and mostpreferably at least 15 amino acids. A peptide which mimics an antigenicfragment of the natural polypeptide may be fused with another proteinsuch as keyhole hemolimpet cyanin (KLH; Sigma, St. Louis, Mo.) forantibody production. A peptide encompassing an antigenic region may beexpressed from an mddt, synthesized as described above, or purified fromhuman cells.

Procedures well known in the art may be used for the production ofantibodies. Various hosts including mice, goats, and rabbits, may beimmunized by injection with a peptide. Depending on the host species,various adjuvants may be used to increase immunological response.

In one procedure, peptides about 15 residues in length may besynthesized using an ABI 431A peptide synthesizer (Applied Biosystems)using fmoc-chemistry and coupled to KLH (Sigma) by reaction withM-maleimidobenzoyl-N-hydroxysuccimide ester (Ausubel, 1995, supra).Rabbits are immunized with the peptide-KLH complex in complete Freund'sadjuvant The resulting antisera are tested for antipeptide activity bybinding the peptide to plastic, blocking with 1% bovine serum albumin(BSA), reacting with rabbit antisera, washing, and reacting withradioiodinated goat anti-rabbit IgG. Antisera with antipeptide activityare tested for anti-MDDT activity using protocols well known in the art,including ELISA, radioimmunoassay (RIA), and immunoblotting.

In another procedure, isolated and purified peptide may be used toimmunize mice (about 100 μg of peptide) or rabbits (about 1 mg ofpeptide). Subsequently, the peptide is radioiodinated and used to screenthe immunized animals' B-lymphocytes for production of antipeptideantibodies. Positive cells are then used to produce hybridomas usingstandard techniques. About 20 mg of peptide is sufficient for labelingand screening several thousand clones. Hybridomas of interest aredetected by screening with radioiodinated peptide to identify thosefusions producing peptide-specific monoclonal antibody. In a typicalprotocol, wells of a multi-well plate (FAST, Becton-Dickinson, PaloAlto, Calif.) are coated with affinity-purified, specificrabbit-anti-mouse (or suitable anti-species IgG) antibodies at 10 mg/ml.The coated wells are blocked with 1% BSA and washed and exposed tosupernatants from hybridomas. After incubation, the wells are exposed toradiolabeled peptide at 1 mg/ml.

Clones producing antibodies bind a quantity of labeled peptide that isdetectable above background. Such clones are expanded and subjected to 2cycles of cloning. Cloned hybridomas are injected into pristane-treatedmice to produce ascites, and monoclonal antibody is purified from theascitic fluid by affinity chromatography on protein A (AmershamPharmacia Biotech). Several procedures for the production of monoclonalantibodies, including in vitro production, are described in Pound(supra). Monoclonal antibodies with antipeptide activity are tested foranti-MDDT activity using protocols well known in the art, includingELISA, RIA, and immunoblotting.

Antibody fragments containing specific binding sites for an epitope mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments produced by pepsin digestion of theantibody molecule, and the Fab fragments generated by reducing thedisulfide bridges of the F(ab′)2 fragments. Alternatively, constructionof Fab expression libraries in filamentous bacteriophage allows rapidand easy identification of monoclonal fragments with desired specificity(Pound, supra, Chaps. 45-47). Antibodies generated against polypeptideencoded by mddt can be used to purify and characterize full-length MDDTprotein and its activity, binding partners, etc.

Assays Using Antibodies

Anti-MDDT antibodies may be used in assays to quantify the amount ofMDDT found in a particular human cell. Such assays include methodsutilizing the antibody and a label to detect expression level undernormal or disease conditions. The peptides and antibodies of theinvention may be used with or without modification or labeled by joiningthem, either covalently or noncovalently, with a reporter molecule.

Protocols for detecting and measuring protein expression using eitherpolyclonal or monoclonal antibodies are well known in the art Examplesinclude ELISA, RIA, and fluorescent activated cell sorting (FACS). Suchimmunoassays typically involve the formation of complexes between theMDDT and its specific antibody and the measurement of such complexes.These and other assays are described in Pound (supra).

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

The disclosures of all patents, applications, and publications mentionedabove and below, in particular U.S. Ser. No. 60/185,213, U.S. Ser. No.60/205,285, U.S. Ser. No. 60/205,232, U.S. Ser. No. 60/205,323, U.S.Ser. No. 60/205,287, U.S. Ser. No. 60/205,324, and U.S. Ser. No.60/205,286, are hereby expressly incorporated by reference.

EXAMPLES

I. Construction of cDNA Libraries

RNA was purchased from CLONTECH Laboratories, Inc. (Palo Alto, Calif.)or isolated from various tissues. Some tissues were homogenized andlysed in guanidinium isothiocyanate, while others were homogenized andlysed in phenol or in a suitable mixture of denaturants, such as TRIZOL(Life Technologies), a monophasic solution of phenol and guanidineisothiocyanate. The resulting lysates were centrifuged over CsClcushions or extracted with chloroform. RNA was precipitated with eitherisopropanol or sodium acetate and ethanol, or by other routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary toincrease RNA purity. In most cases, RNA was treated with DNase. For mostlibraries, poly(A+) RNA was isolated using oligo d(T)-coupledparamagnetic particles (Promega Corporation (Promega), Madison, Wis.),OLIGOTEX latex particles (QIAGEN, Inc. (QIAGEN), Valencia, Calif.), oran OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA wasisolated directly from tissue lysates using other RNA isolation kits,e.g., the POLY(A)PURE mRNA purification kit (Ambion, Inc., Austin,Tex.).

In some cases, Stratagene was provided with RNA and constructed thecorresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNAlibraries were constructed with the UNIZAP vector system (StratageneCloning Systems, Inc. (Stratagene), La Jolla, Calif.) or SUPERSCRIPTplasmid system (Life Technologies), using the recommended procedures orsimilar methods known in the art. (See, e.g., Ausubel, 1997, supra,Chapters 5.1 through 6.6.) Reverse transcription was initiated usingoligo d(T) or random primers. Synthetic oligonucleotide adapters wereligated to double stranded cDNA, and the cDNA was digested with theappropriate restriction enzyme or enzymes. For most libraries, the cDNAwas size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B,or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) orpreparative agarose gel electrophoresis. cDNAs were ligated intocompatible restriction enzyme sites of the polylinker of a suitableplasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (LifeTechnologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad, Calif.), PBK-CMVplasmid (Stratagene), or pINCY (Incyte Genomics, Palo Alto, Calif.), orderivatives thereof. Recombinant plasmids were transformed intocompetent E. coli cells including XL1-Blue, XL1-BlueMRF, or SOLR fromStratagene or DH5α, DH10B, or ElectroMAX DH10B from Life Technologies.

II. Isolation of cDNA Clones

Plasmids were recovered from host cells by in vivo excision using theUNIZAP vector system (Stratagene) or by cell lysis. Plasmids werepurified using at least one of the following: the Magic or WIZARDMinipreps DNA purification system (Promega); the AGTC Minipreppurification kit (Edge BioSystems, Gaithersburg, Md.); and the QIAWELL8, QIAWELL 8 Plus, and QIAWELL 8 Ultra plasmid purification systems orthe R.E.A.L. PREP 96 plasmid purification kit (QIAGEN). Followingprecipitation, plasmids were resuspended in 0.1 ml of distilled waterand stored, with or without lyophilization, at 4° C.

Alternatively, plasmid DNA was amplified from host cell lysates usingdirect link PCR in a high-throughput format. (Rao, V. B. (1994) Anal.Biochem. 216:1-14.) Host cell lysis and thermal cycling steps werecarried out in a single reaction mixture. Samples were processed andstored in 384-well plates, and the concentration of amplified plasmidDNA was quantified fluorometrically using PICOGREEN dye (MolecularProbes, Inc. (Molecular Probes), Eugene, Oreg.) and a FLUOROSKAN 11fluorescence scanner (Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

cDNA sequencing reactions were processed using standard methods orhigh-throughput instrumentation such as the ABI CATALYST 800 thermalcycler (Applied Biosystems) or the PTC-200 thermal cycler (MJ Research)in conjunction with the HYDRA microdispenser (Robbins Scientific Corp.,Sunnyvale, Calif.) or the MICROLAB 2200 liquid transfer system(Hamilton). cDNA sequencing reactions were prepared using reagentsprovided by Amersham Pharmacia Biotech or supplied in ABI sequencingkits such as the ABI PRISM BIGDYE Terminator cycle sequencing readyreaction kit (Applied Biosystems). Electrophoretic separation of cDNAsequencing reactions and detection of labeled polynucleotides werecarried out using the MEGABACE 1000 DNA sequencing system (MolecularDynamics); the ABI PRISM 373 or 377 sequencing system (AppliedBiosystems) in conjunction with standard ABI protocols and base callingsoftware; or other sequence analysis systems known in the art. Readingframes within the cDNA sequences were identified using standard methods(reviewed in Ausubel, 1997, supra, Chapter 7.7). Some of the cDNAsequences were selected for extension using the techniques disclosed inExample VIII.

IV. Assembly and Analysis of Sequences

Component sequences from chromatograms were subject to PHRED analysisand assigned a quality score. The sequences having at least a requiredquality score were subject to various pre-processing editing pathways toeliminate, e.g., low quality 3′ ends, vector and linker sequences, polyAtails, Alu repeats, mitochondrial and ribosomal sequences, bacterialcontamination sequences, and sequences smaller than 50 base pairs. Inparticular, low-information sequences and repetitive elements (e.g.,dinucleotide repeats, Alu repeats, etc.) were replaced by “n's”, ormasked, to prevent spurious matches.

Processed sequences were then subject to assembly procedures in whichthe sequences were assigned to gene bins (bins). Each sequence couldonly belong to one bin. Sequences in each gene bin were assembled toproduce consensus sequences (templates). Subsequent new sequences wereadded to existing bins using BLASTn (v.1.4 WashU) and CROSSMATCH.Candidate pairs were identified as all BLAST hits having a quality scoregreater than or equal to 150. Alignments of at least 82% local identitywere accepted into the bin. The component sequences from each bin wereassembled using a version of PHRAP. Bins with several overlappingcomponent sequences were assembled using DEEP PHAP. The orientation(sense or antisense) of each assembled template was determined based onthe number and orientation of its component sequences. Templatesequences as disclosed in the sequence listing correspond to sensestrand sequences (the “forward” reading frames), to the bestdetermination. The complementary (antisense) strands are inherentlydisclosed herein. The component sequences which were used to assembleeach template consensus sequence are listed in Table 4, along with theirpositions along the template nucleotide sequences.

Bins were compared against each other and those having local similarityof at least 82% were combined and reassembled. Reassembled bins havingtemplates of insufficient overlap (less than 95% local identity) werere-split. Assembled templates were also subject to analysis bySTITCHER/EXON MAPPER algorithms which analyze the probabilities of thepresence of splice variants, alternatively spliced exons, splicejunctions, differential expression of alternative spliced genes acrosstissue types or disease states, etc. These resulting bins were subjectto several rounds of the above assembly procedures.

Once gene bins were generated based upon sequence alignments, bins wereclone joined based upon clone information. If the 5′ sequence of oneclone was present in one bin and the 3′ sequence from the same clone waspresent in a different bin, it was likely that the two bins actuallybelonged together in a single bin. The resulting combined bins underwentassembly procedures to regenerate the consensus sequences.

The final assembled templates were subsequently annotated using thefollowing procedure. Template sequences were analyzed using BLASTn(v2.0, NCBI) versus gbpri (GenBank version 120). “Hits” were defined asan exact match having from 95% local identity over 200 base pairsthrough 100% local identity over 100 base pairs, or a homolog matchhaving an E-value, i.e. a probability score, of ≦1×10⁻⁸. The hits weresubject to frameshift FASTx versus GENPEPT (GenBank version 120). (SeeTable 7). In this analysis, a homolog match was defined as having anE-value of ≦1×10⁻⁸. The assembly method used above was described in“System and Methods for Analyzing Biomolecular Sequences,” U.S. Ser. No.09/276,534, filed Mar. 25, 1999, and the LIFESEQ Gold user manual(Incyte) both incorporated by reference herein.

Following assembly, template sequences were subjected to motif, BLAST,and functional analyses, and categorized in protein hierarchies usingmethods described in, e.g., “Database System Employing Protein FunctionHierarchies for Viewing Biomolecular Sequence Data,” U.S. Ser. No.08/812,290, filed Mar. 6, 1997; “Relational Database for StoringBiomolecule Information,” U.S. Ser. No. 08/947,845, filed Oct. 9, 1997;“Project-Based Full-Length Biomolecular Sequence Database,” U.S. Ser.No. 08/811,758, filed Mar. 6, 1997; and “Relational Database and Systemfor Storing Information Relating to Biomolecular Sequences,” U.S. Ser.No. 09/034,807, filed Mar. 4, 1998, all of which are incorporated byreference herein.

The template sequences were further analyzed by translating eachtemplate in all three forward reading frames and searching eachtranslation against the Pfam database of hidden Markov model-basedprotein families and domains using the HMMER software package (availableto the public from Washington University School of Medicine, St Louis,Mo.). Regions of templates which, when translated, contain similarity toPfam consensus sequences are reported in Table 2, along withdescriptions of Pfam protein domains and families. Only those Pfam hitswith an E-value of ≦1×10⁻³ are reported. (See also World Wide Web sitehttp://pfam.wustl.edu/ for detailed descriptions of Pfam protein domainsand families.)

Additionally, the template sequences were translated in all threeforward reading frames, and each translation was searched against hiddenMarkov models for signal peptides using the HMMER software package.Construction of hidden Markov models and their usage in sequenceanalysis has been described. (See, for example, Eddy, S. R. (1996) Curr.Opin. Str. Biol. 6:361-365.) Only those signal peptide hits with acutoff score of 11 bits or greater are reported. A cutoff score of 11bits or greater corresponds to at least about 91-94% true-positives insignal peptide prediction. Template sequences were also translated inall three forward reading frames, and each translation was searchedagainst TMAP, a program that uses weight matrices to delineatetransmembrane segments on protein sequences and determine orientation,with respect to the cell cytosol (Persson, B. and P. Argos (1994) J.Mol. Biol. 237:182-192; Persson, B. and P. Argos (1996) Protein Sci.5:363-371.) Regions of templates which, when translated, containsimilarity to signal peptide or transmembrane consensus sequences arereported in Table 3.

The results of HMMER analysis as reported in Tables 2 and 3 may supportthe results of BLAST analysis as reported in Table 1 or may suggestalternative or additional properties of template-encoded polypeptidesnot previously uncovered by BLAST or other analyses.

Template sequences are further analyzed using the bioinformatics toolslisted in Table 7, or using sequence analysis software known in the artsuch as MACDNASIS PRO software (Hitachi Software Engineering, South SanFrancisco, Calif.) and LASERGENE software (DNASTAR). Template sequencesmay be further queried against public databases such as the GenBankrodent, mammalian, vertebrate, prokaryote, and eukaryote databases.

The template sequences were translated to derive the correspondinglongest open reading frame as presented by the polypeptide sequences.Alternatively, a polypeptide of the invention may begin at any of themethionine residues within the full length translated polypeptide.Polypeptide sequences were subsequently analyzed by querying against theGenBank protein database (GENPEPT, (GenBank version 121)). Full lengthpolynucleotide sequences are also analyzed using MACDNASIS PRO software(Hitachi Software Engineering, South San Francisco, Calif.) andLASERGENE software (DNASTAR). Polynucleotide and polypeptide sequencealignments are generated using default parameters specified by theCLUSTAL algorithm as incorporated into the MEGALIGN multisequencealignment program (DNASTAR), which also calculates the percent identitybetween aligned sequences.

Table 6 shows sequences with homology to the polypeptides of theinvention as identified by BLAST analysis against the GenBank protein(GENPEPT) database. Column 1 shows the polypeptide sequenceidentification number (SEQ ID NO:) for the polypeptide segments of theinvention. Column 2 shows the reading frame used in the translation ofthe polynucleotide sequences encoding the polypeptide segments. Column 3shows the length of the translated polypeptide segments. Columns 4 and 5show the start and stop nucleotide positions of the polynucleotidesequences encoding the polypeptide segments. Column 6 shows the GenBankidentification number (GI Number) of the nearest GenBank homolog. Column7 shows the probability score for the match between each polypeptide andits GenBank homolog. Column 8 shows the annotation of the GenBankhomolog.

V. Analysis of Polynucleotide Expression

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7;Ausubel, 1995, supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST were used to search foridentical or related molecules in cDNA databases such as GenBank orLIFESEQ (Incyte Genomics). This analysis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score, which is defined as:$\frac{{BLAST}\quad{Score} \times {Percent}\quad{Identity}}{5 \times {minimum}\quad\left\{ {{{length}\quad\left( {{Seq}.\quad 1} \right)},{{length}\left( {{Seq}.\quad 2} \right)}} \right\}}$The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match The productscore is a normalized value between 0 and 100, and is calculated asfollows: the BLAST score is multiplied by the percent nucleotideidentity and the product is divided by (5 times the length of theshorter of the two sequences). The BLAST score is calculated byassigning a score of +5 for every base that matches in a high-scoringsegment pair (HSP), and −4 for every mismatch. Two sequences may sharemore than one HSP (separated by gaps). If there is more than one HSP,then the pair with the highest BLAST score is used to calculate theproduct score. The product score represents a balance between fractionaloverlap and quality in a BLAST alignment. For example, a product scoreof 100 is produced only for 100% identity over the entire length of theshorter of the two sequences being compared. A product score of 70 isproduced either by 100% identity and 70% overlap at one end, or by 88%identity and 100% overlap at the other. A product score of 50 isproduced either by 100% identity and 50% overlap at one end, or 79%identity and 100% overlap.

VI. Tissue Distribution Profiling

A tissue distribution profile is determined for each template bycompiling the cDNA library tissue classifications of its component cDNAsequences. Each component sequence, is derived from a cDNA libraryconstructed from a human tissue. Each human tissue is classified intoone of the following categories: cardiovascular system; connectivetissue; digestive system; embryonic structures; endocrine system;exocrine glands; genitalia, female; genitalia, male; germ cells; hemicand immune system; liver; musculoskeletal system; nervous system;pancreas; respiratory system; sense organs; skin; stomatognathic system;unclassified/mixed; or urinary tract. Template sequences, componentsequences, and cDNA library/tissue information are found in the LIFESEQGOLD database (Incyte Genomics, Palo Alto, Calif.).

Table 5 shows the tissue distribution profile for the templates of theinvention. For each template, the three most frequently observed tissuecategories are shown in column 3, along with the percentage of componentsequences belonging to each category. Only tissue categories withpercentage values of ≧10% are shown. A tissue distribution of “widelydistributed” in column 3 indicates percentage values of <10% in alltissue categories.

VII. Transcript Image Analysis

Transcript images are generated as described in Seilhamer et al.,“Comparative Gene Transcript Analysis,” U.S. Pat. No. 5,840,484,incorporated herein by reference.

VIII. Extension of Polynucleotide Sequences and Isolation of aFull-length cDNA

Oligonucleotide primers designed using an mddt of the Sequence Listingare used to extend the nucleic acid sequence. One primer is synthesizedto initiate 5′ extension of the template, and the other primer, toinitiate 3′ extension of the template. The initial primers may bedesigned using OLIGO 4.06 software (National Biosciences, Inc. (NationalBiosciences), Plymouth, Minn.), or another appropriate program, to beabout 22 to 30 nucleotides in length, to have a GC content of about 50%or more, and to anneal to the target sequence at temperatures of about68° C. to about 72° C. Any stretch of nucleotides which would result inhairpin structures and primer-primer dimerizations are avoided. Selectedhuman cDNA libraries are used to extend the sequence. If more than oneextension is necessary or desired, additional or nested sets of primersare designed.

High fidelity amplification is obtained by PCR using methods well knownin the art. PCR is performed in 96-well plates using the PTC-200 thermalcycler (MJ Research). The reaction mix contains DNA template, 200 nmolof each primer, reaction buffer containing Me²⁺, (NH₄)₂SO₄, andβ-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase(Stratagene), with the following parameters for primer pair PCI A andPCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, theparameters for primer pair T7 and SK+ are as follows: Step 1: 94° C., 3min; Step 2:

to determine which reactions are successful in extending the sequence.

The extended nucleotides are desalted and concentrated, transferred to384-well plates, digested with CviJI cholera virus endonuclease(Molecular Biology Research, Madison, Wis.), and sonicated or shearedprior to religation into pUC 18 vector (Amersham Pharmacia Biotech). Forshotgun sequencing, the digested nucleotides are separated on lowconcentration (0.6 to 0.8%) agarose gels, fragments are excised, andagar digested with AGAR ACE (Promega). Extended clones are religatedusing T4 ligase (New England Biolabs, Inc., Beverly, Mass.) into pUC 18vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase(Stratagene) to fill-in restriction site overhangs, and transfected intocompetent E. coli cells. Transformed cells are selected onantibiotic-containing media, individual colonies are picked and culturedovernight at 37° C. in 384-well plates in LB/2× carbenicillin liquidmedia.

The cells are lysed, and DNA is amplified by PCR using Taq DNApolymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase(Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5:steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7:storage at 4° C. DNA is quantified by PICOGREEN reagent (MolecularProbes) as described above. Samples with low DNA recoveries arereamplified using the same conditions as described above. Samples arediluted with 20% dimethysulfoxide (1:2, v/v), and sequenced usingDYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cyclesequencing ready reaction kit (Applied Biosystems).

In like manner, the mddt is used to obtain regulatory sequences(promoters, introns, and enhancers) using the procedure above,oligonucleotides designed for such extension, and an appropriate genomiclibrary.

IX. Labeling of Probes and Southern Hybridization Analyses

Hybridization probes derived from the mddt of the Sequence Listing areemployed for screening cDNAs, mRNAs, or genomic DNA. The labeling ofprobe nucleotides between 100 and 1000 nucleotides in length isspecifically described, but essentially the same procedure may be usedwith larger cDNA fragments. Probe sequences are labeled at roomtemperature for 30 minutes using a T4 polynucleotide kinase, γ²P-ATP,and 0.5× One-Phor-All Plus (Amersham Pharmacia Biotech) buffer andpurified using a ProbeQuant G-50 Microcolumn (Amersham PharmaciaBiotech). The probe mixture is diluted to 10⁷ dpm/μg/ml hybridizationbuffer and used in a typical membrane-based hybridization analysis.

The DNA is digested with a restriction endonuclease such as Eco RV andis electrophoresed through a 0.7% agarose gel. The DNA fragments aretransferred from the agarose to nylon membrane (NYTRAN Plus, Schleicher& Schuell, Inc., Keene, N.H.) using procedures specified by themanufacturer of the membrane. Prehybridization is carried out for threeor more hours at 68° C., and hybridization is carried out overnight at68° C. To remove non-specific signals, blots are sequentially washed atroom temperature under increasingly stringent conditions, up to 0.1×saline sodium citrate (SSC) and 0.5% sodium dodecyl sulfate. After theblots are placed in a PHOSPHORIMAGER cassette (Molecular Dynamics) orare exposed to autoradiography film, hybridization patterns of standardand experimental lanes are compared. Essentially the same procedure isemployed when screening RNA.

X. Chromosome Mapping of mddt

The cDNA sequences which were used to assemble SEQ ID NO:1-45 arecompared with sequences from the Incyte LIFESEQ database and publicdomain databases using BLAST and other implementations of theSmith-Waterman algorithm. Sequences from these databases that match SEQID NO:1-45 are assembled into clusters of contiguous and overlappingsequences using assembly algorithms such as PHRAP (Table 7). Radiationhybrid and genetic mapping data available from public resources such asthe Stanford Human Genome Center (SHGC), Whitehead Institute for GenomeResearch (WIGR), and Généthon are used to determine if any of theclustered sequences have been previously mapped. Inclusion of a mappedsequence in a cluster will result in the assignment of all sequences ofthat cluster, including its particular SEQ ID NO:, to that map location.The genetic map locations of SEQ ID NO:1-45 are described as ranges, orintervals, of human chromosomes. The map position of an interval, incentiMorgans, is measured relative to the terminus of the chromosome'sp-arm. (The centiMorgan (cM) is a unit of measurement based onrecombination frequencies between chromosomal markers. On average, 1 cMis roughly equivalent to 1 megabase (Mb) of DNA in humans, although thiscan vary widely due to hot and cold spots of recombination.) The cMdistances are based on genetic markers mapped by Généthon which provideboundaries for radiation hybrid markers whose sequences were included ineach of the clusters.

XI. Microarray Analysis

Probe Preparation from Tissue or Cell Samples

Total RNA is isolated from tissue samples using the guanidiniumthiocyanate method and polyA⁺ RNA is purified using the oligo (dT)cellulose method. Each polyA⁺ RNA sample is reverse transcribed usingMMLV reverse-transcriptase, 0.05 pg/l oligo-T primer (21 mer), 1× firststrand buffer, 0.03 units/μl RNase inhibitor, 500 μM DATP, 500 μM dGTP,500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (AmershamPharmacia Biotech). The reverse transcription reaction is performed in a25 ml volume containing 200 ng polyA⁺ RNA with GEMBRIGHT kits (Incyte).Specific control polyA⁺ RNAs are synthesized by in vitro transcriptionfrom non-coding yeast genomic DNA (W. Lei, unpublished). As quantitativecontrols, the control mRNAs at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng arediluted into reverse transcription reaction at ratios of 1:100,000,1:10,000, 1:1000, 1:100 (w/w) to sample mRNA respectively. The controlmRNAs are diluted into reverse transcription reaction at ratios of 1:3,3:1, 1:10, 10:1, 1:25, 25:1 (w/w) to sample/mRNA differential expressionpatterns. After incubation at 37° C. for 2 hr, each reaction sample (onewith Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5Msodium hydroxide and incubated for 20 minutes at 85° C. to the stop thereaction and degrade the RNA. Probes are purified using two successiveCHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.(CLONTECH), Palo Alto, Calif.) and after combining, both reactionsamples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 mlsodium acetate, and 300 ml of 100% ethanol. The probe is then dried tocompletion using a SpeedVAC (Savant Instruments Inc., Holbrook, N.Y.)and resuspended in 14 μl 5×SSC/0.2% SDS.

Microarray Preparation

Sequences of the present invention are used to generate array elements.Each array element is amplified from bacterial cells containing vectorswith cloned cDNA inserts. PCR amplification uses primers complementaryto the vector sequences flanking the cDNA insert Array elements areamplified in thirty cycles of PCR from an initial quantity of 1-2 ng toa final quantity greater than 5 μg. Amplified array elements are thenpurified using SEPHACRYL-400 (Amersham Pharmacia Biotech).

Purified array elements are immobilized on polymer-coated glass slides.Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDSand acetone, with extensive distilled water washes between and aftertreatments. Glass slides are etched in 4% hydrofluoric acid (VWRScientific Products Corporation (VWR), West Chester, Pa.), washedextensively in distilled water, and coated with 0.05% aminopropyl silane(Sigma) in 95% ethanol. Coated slides are cured in a 110° C. oven.

Array elements are applied to the coated glass substrate using aprocedure described in U.S. Pat. No. 5,807,522, incorporated herein byreference. 1 μl of the array element DNA, at an average concentration of100 ng/l, is loaded into the open capillary printing element by ahigh-speed robotic apparatus. The apparatus then deposits about 5 nl ofarray element sample per slide.

Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker(Stratagene). Microarrays are washed at room temperature once in 0.2%SDS and three times in distilled water. Non-specific binding sites areblocked by incubation of microarrays in 0.2% casein in phosphatebuffered saline (PBS) (Tropix, Inc., Bedford, Mass.) for 30 minutes at60° C. followed by washes in 0.2% SDS and distilled water as before.

Hybridization

Hybridization reactions contain 9 μl of probe mixture consisting of 0.2μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2%SDS hybridization buffer. The probe mixture is heated to 65° C. for 5minutes and is aliquoted onto the microarray surface and covered with an1.8 cm² coverslip. The arrays are transferred to a waterproof chamberhaving a cavity just slightly larger than a microscope slide. Thechamber is kept at 100% humidity internally by the addition of 140 μl of5×SSC in a corner of the chamber. The chamber containing the arrays isincubated for about 6.5 hours at 60° C. The arrays are washed for 10 minat 45° C. in a first wash buffer (1×SSC, 0.1% SDS), three times for 10minutes each at 45° C. in a second wash buffer (0.1×SSC), and dried.

Detection

Reporter-labeled hybridization complexes are detected with a microscopeequipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., SantaClara, Calif.) capable of generating spectral lines at 488 nm forexcitation of Cy3 and at 632 nm for excitation of Cy5. The excitationlaser light is focused on the array using a 20× microscope objective(Nikon, Inc., Melville, N.Y.). The slide containing the array is placedon a computer-controlled X-Y stage on the microscope and raster-scannedpast the objective. The 1.8 cm×1.8 cm array used in the present exampleis scanned with a resolution of 20 micrometers.

In two separate scans, a mixed gas multiline laser excites the twofluorophores sequentially. Emitted light is split, based on wavelength,into two photomultiplier tube detectors (PMT R1477, Hamamatsu PhotonicsSystems, Bridgewater, N.J.) corresponding to the two fluorophores.Appropriate filters positioned between the array and the photomultipliertubes are used to filter the signals. The emission maxima of thefluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array istypically scanned twice, one scan per fluorophore using the appropriatefilters at the laser source, although the apparatus is capable ofrecording the spectra from both fluorophores simultaneously.

The sensitivity of the scans is typically calibrated using the signalintensity generated by a cDNA control species added to the probe mix ata known concentration A specific location on the array contains acomplementary DNA sequence, allowing the intensity of the signal at thatlocation to be correlated with a weight ratio of hybridizing species of1:100,000. When two probes from different sources (e.g., representingtest and control cells), each labeled with a different fluorophore, arehybridized to a single array for the purpose of identifying genes thatare differentially expressed, the calibration is done by labelingsamples of the calibrating cDNA with the two fluorophores and addingidentical amounts of each to the hybridization mixture.

The output of the photomultiplier tube is digitized using a 12-bitRTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc.,Norwood, Mass.) installed in an IBM-compatible PC computer. Thedigitized data are displayed as an image where the signal intensity ismapped using a linear 20-color transformation to a pseudocolor scaleranging from blue (low signal) to red (high signal). The data is alsoanalyzed quantitatively. Where two different fluorophores are excitedand measured simultaneously, the data are first corrected for opticalcrosstalk (due to overlapping emission spectra) between the fluorophoresusing each fluorophore's emission spectrum.

A grid is superimposed over the fluorescence signal image such that thesignal from each spot is centered in each element of the grid. Thefluorescence signal within each element is then integrated to obtain anumerical value corresponding to the average intensity of the signal.The software used for signal analysis is the GEMTOOLS gene expressionanalysis program (Incyte).

XII. Complementary Nucleic Acids

Sequences complementary to the mddt are used to detect, decrease, orinhibit expression of the naturally occurring nucleotide. The use ofoligonucleotides comprising from about 15 to 30 base pairs is typical inthe art. However, smaller or larger sequence fragments can also be used.Appropriate oligonucleotides are designed from the mddt using OLIGO 4.06software (National Biosciences) or other appropriate programs and aresynthesized using methods standard in the art or ordered from acommercial supplier. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent transcription factor binding to the promoter sequence. Toinhibit translation, a complementary oligonucleotide is designed toprevent ribosomal binding and processing of the transcript.

XIII. Expression of MDDT

Expression and purification of MDDT is accomplished using bacterial orvirus-based expression systems. For expression of MDDT in bacteria, cDNAis subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express MDDT uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof MDDT in eukaryotic cells is achieved by infecting insect or mammaliancell lines with recombinant Autographica califonica nuclear polyhedrosisvirus (AcMNPV), commonly known as baculovirus. The nonessentialpolyhedrin gene of baculovirus is replaced with cDNA encoding MDDT byeither homologous recombination or bacterial-mediated transpositioninvolving transfer plasmid intermediates. Viral infectivity ismaintained and the strong polyhedrin promoter drives high levels of cDNAtranscription. Recombinant baculovirus is used to infect Spodopterafrugiperda (Sf9) insect cells in most cases, or human hepatocytes, insome cases. Infection of the latter requires additional geneticmodifications to baculovirus. (See e.g., Engelhard, supra; and Sandig,supra.)

In most expression systems, MDDT is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (AmershamPharmacia Biotech). Following purification, the GST moiety can beproteolytically cleaved from MDDT at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak Company, Rochester, N.Y.). 6-His, a stretch of sixconsecutive histidine residues, enables purification on metal-chelateresins (QIAGEN). Methods for protein expression and purification arediscussed in Ausubel (1995, supra, Chapters 10 and 16). Purified MDDTobtained by these methods can be used directly in the following activityassay.

XIV. Demonstration of MDDT Activity

MDDT, or biologically active fragments thereof, are labeled with ¹²⁵IBolton-Hunter reagent. (See, e.g., Bolton, A. E. and W. M. Hunter (1973)Biochem. J. 133:529-539.) Candidate molecules previously arrayed in thewells of a multi-well plate are incubated with the labeled MDDT, washed,and any wells with labeled MDDT complex are assayed. Data obtained usingdifferent concentrations of MDDT are used to calculate values for thenumber, affinity, and association of MDDT with the candidate molecules.

Alternatively, molecules interacting with MDDT are analyzed using theyeast two-hybrid system as described in Fields, S. and O. Song (1989)Nature 340:245-246, or using commercially available kits based on thetwo-hybrid system, such as the MATCHMAKER system (CLONTECH).

MDDT may also be used in the PATHCALLING process (CuraGen Corp., NewHaven, Conn.) which employs the yeast two-hybrid system in ahigh-throughput manner to determine all interactions between theproteins encoded by two large libraries of genes (Nandabalan, K. et al.(2000) U.S. Pat. No. 6,057,101).

XV. Functional Assays

MDDT function is assessed by expressing mddt at physiologically elevatedlevels in mammalian cell culture systems. cDNA is subcloned into amammalian expression vector containing a strong promoter that driveshigh levels of cDNA expression Vectors of choice include pCMV SPORT(Life Technologies) and pCR3.1 (Invitrogen Corporation, Carlsbad,Calif.), both of which contain the cytomegalovirus promoter. 5-10 μg ofrecombinant vector are transiently transfected into a human cell line,preferably of endothelial or hematopoietic origin, using either liposomeformulations or electroporation. 1-2 μg of an additional plasmidcontaining sequences encoding a marker protein are co-transfected.

Expression of a marker protein provides a means to distinguishtransfected cells from nontransfected cells and is a reliable predictorof cDNA expression from the recombinant vector. Marker proteins ofchoice include, e.g., Green Fluorescent Protein (GFP; CLONTECH), CD64,or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated laseroptics-based technique, is used to identify transfected cells expressingGFP or CD64-GFP and to evaluate the apoptotic state of the cells andother cellular properties.

FCM detects and quantifies the uptake of fluorescent molecules thatdiagnose events preceding or coincident with cell death These eventsinclude changes in nuclear DNA content as measured by staining of DNAwith propidium iodide; changes in cell size and granularity as measuredby forward light scatter and 90 degree side light scatter;down-regulation of DNA synthesis as measured by decrease inbromodeoxyuridine uptake; alterations in expression of cell surface andintracellular proteins as measured by reactivity with specificantibodies; and alterations in plasma membrane composition as measuredby the binding of fluorescein-conjugated Annexin V protein to the cellsurface. Methods in flow cytometry are discussed in Ormerod, M. G.(1994) Flow Cytometry, Oxford, New York, N.Y.

The influence of MDDT on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding MDDTand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either ban IgG orantibody against CD64 (DYNAL, Inc., Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art Expression of mRNA encoding MDDT and other genes of interest canbe analyzed by northern analysis or microarray techniques.

XVI. Production of Antibodies

MDDT substantially purified using polyacrylamide gel electrophoresis(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the MDDT amino acid sequence is analyzed using LASERGENEsoftware (DNASTAR) to determine regions of high immunogenicity, and acorresponding peptide is synthesized and used to raise antibodies bymeans known to those of skill in the art Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art. (See, e.g., Ausubel,1995, supra, Chapter 11.)

Typically, peptides 15 residues in length are synthesized using an ABI431A peptide synthesizer (Applied Biosystems) using fmoc-chemistry andcoupled to KLH (Sigma) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel, supra.) Rabbits are immunized withthe peptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity by, for example, bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radii iodinated goat anti-rabbitIgG. Antisera with antipeptide activity are tested for anti-MDDT,activity using protocols well known in the art, including ELISA, RIA,and immunoblotting.

XVII. Purification of Naturally Occurring MDDT Using Specific Antibodies

Naturally occurring or recombinant MDDT is substantially purified byimmunoaffinity chromatography using antibodies specific for MDDT. Animmunoaffinity column is constructed by covalently coupling anti-MDDTantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing MDDT are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof MDDT (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/MDDT binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and MDDTis collected.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims. TABLE 1 SEQ ID Probability NO:Template ID GI Number Score Annotation 1 LG:977683.1:2000FEB18 g107647780 phosphoinositol 3-phosphate-binding protein-2 (Homo 2LG:893050.1:2000FEB18 g6634025 2.00E−81 KIAA0379 protein (Homo sapiens)3 LG:980153.1:2000FEB18 g7263990 0 dJ93K22.1 (novel protein (containsDKFZP564B116)) (Homo sapiens) 4 LG:350398.1:2000FEB18 g3882175 3.00E−10KIAA0727 protein (Homo sapiens) 5 LG:475551.1:2000FEB18 g861029 0 SH3domain binding protein (Mus musculus) 6 LG:481407.2:2000FEB18 g61195461.00E−41 hypothetical protein; 114721-113936 (Arabidopsis thaliana) 7LI:443580.1:2000FEB01 g4589566 3.00E−34 KIAA0961 protein (Homo sapiens)8 LI:803015.1:2000FEB01 g5262560 2.00E−35 hypothetical protein (Homosapiens) 9 LG:027410.3:2000MAY19 g10438267 1.00E−65 unnamed proteinproduct (Homo sapiens) 10 LG:171377.1:2000MAY19 g3077703 1.00E−107mitsugumin29 (Oryctolagus cuniculus) 11 LG:352559.1:2000MAY19 g72432432.00E−43 KIAA1431 protein (Homo sapiens) 12 LG:247384.1:2000MAY19g9945010 1.00E−118 RING-finger protein MURF (Mus musculus) 13LG:403872.1:2000MAY19 g7020303 0 unnamed protein product (Homo sapiens)14 LG:1135213.1:2000MAY19 g6692607 2.00E−65 MGA protein (Mus musculus)15 LG:474284.2:2000MAY19 g1488047 2.00E−30 RING finger protein (Xenopuslaevis) 16 LG:342147.1:2000MAY19 g2477511 3.00E−41 Homo sapiens p20protein (pir B53814) 17 LG:1097300.1:2000MAY19 g2078531 1.00E−70 Mlark(Mus musculus) 18 LG:444850.9:2000MAY19 g199000 0 interferon-gammainducible protein (Mus musculus) 19 LG:402231.6:2000MAY19 g70207376.00E−77 unnamed protein product (Homo sapiens) 20LG:1076157.1:2000MAY19 g5262560 3.00E−65 hypothetical protein (Homosapiens) 21 LG:1083142.1:2000MAY19 g4589566 3.00E−23 KIAA0961 protein(Homo sapiens) 22 LG:1083264.1:2000MAY19 g10047297 2.00E−25 KIAA1611protein (Homo sapiens) 23 LG:350793.2:2000MAY19 g7242973 0 KIAA1309protein (Homo sapiens) 24 LG:408751.3:2000MAY19 g8886025 1.00E−134collapsin response mediator protein-5 (Homo sapiens) 25LI:336120.1:2000MAY01 g1864085 1.00E−160 glypican-5 (Homo sapiens) 26LI:234104.2:2000MAY01 g1518505 1.00E−114 G-protein coupled inwardlyrectifying K+ channel (Mus musculus) 27 LI:450887.1:2000MAY01 g76299943.00E−34 60S RIBOSOMAL PROTEIN L36 homolog (Arabidopsis thaliana) 28LI:119992.3:2000MAY01 g7243089 0 KIAA1354 protein (Homo sapiens) 29LI:197241.2:2000MAY01 g7263990 0 dJ93K22.1 (novel protein (containsDKFZP564B116)) (Homo sapiens) 30 LI:406860.20:2000MAY01 g104359193.00E−57 unnamed protein product (Homo sapiens) 31 LI:142384.1:2000MAY01g10436290 1.00E−131 unnamed protein product (Homo sapiens) 32LI:895427.1:2000MAY01 g3184264 1.00E−106 F02569_2 (Homo sapiens) 33LI:757439.1:2000MAY01 g7670362 1.00E−116 unnamed protein product (Musmusculus) 34 LI:1144066.1:2000MAY01 g3882281 7.00E−79 KIAA0780 protein(Homo sapiens) 35 LI:243660.4:2000MAY01 g4210501 0 BC85722_1 (Homosapiens) 36 LI:334386.1:2000MAY01 g6330617 0 KIAA1223 protein (Homosapiens) 37 LI:347572.1:2000MAY01 g9802433 1.00E−101 ACE-relatedcarboxypeptidase ACE2 (Homo sapiens) 38 LI:817314.1:2000MAY01 g5802615 0transient receptor potential 4 (Homo sapiens) 39 LI:000290.1:2000MAY01g7242977 2.00E−51 KIAA1311 protein (Homo sapiens) 40LI:023518.3:2000MAY01 g736727 2.00E−74 32 kd accessory protein (Bostaurus) 41 LI:1084246.1:2000MAY01 g5457031 0 protocadherin beta 12 (Homosapiens) 42 LI:1165828.1:2000MAY01 g5457019 0 protocadherin alpha 7short form protein (Homo sapiens) 43 LI:007302.1:2000MAY01 g5006250 0TLR6 (Mus musculus) 44 LI:236386.4:2000MAY01 g6164628 1.00E−63 SH3 andPX domain-containing protein SH3PX1 (Homo sapiens) 45LI:252904.5:2000MAY01 g7022971 2.00E−62 unnamed protein product (Homosapiens)

TABLE 2 SEQ ID NO: Template ID Start Stop Frame Pfam Hit PfamDescription E-value 1 LG:977683.1:2000FEB18 540 695 forward 3 PH PHdomain 6.70E−11 1 LG:977683.1:2000FEB18 204 293 forward 3 WW WW domain7.50E−05 2 LG:893050.1:2000FEB18 211 309 forward 1 ank Ank repeat1.60E−05 3 LG:980153.1:2000FEB18 754 852 forward 1 ank Ank repeat8.00E−04 3 LG:980153.1:2000FEB18 2131 2565 forward 1 BTB BTB/POZ domain6.90E−07 3 LG:980153.1:2000FEB18 1084 1239 forward 1 RCC1 Regulator ofchromosome condensation 3.70E−04 4 LG:350398.1:2000FEB18 7 123 forward 1myosin_head Myosin head (motor domain) 2.60E−16 5 LG:475551.1:2000FEB18702 1157 forward 3 RhoGAP RhoGAP domain 8.10E−71 6 LG:481407.2:2000FEB18225 440 forward 3 rrm RNA recognition motif. (a.k.a. RRM, RBC 1.50E−22 6LG:481407.2:2000FEB18 504 557 forward 3 zf-CCHC Zinc knuckle 7.00E−04 7LI:443580.1:2000FEB01 262 450 forward 1 KRAB KRAB box 1.60E−41 7LI:443580.1:2000FEB01 625 693 forward 1 zf-C2H2 Zinc finger, C2H2 type2.20E−06 8 LI:803015.1:2000FEB01 159 299 forward 3 KRAB KRAB box2.30E−17 9 LG:027410.3:2000MAY19 177 290 forward 3 WD40 WD domain,G-beta repeat 6.20E−06 10 LG:171377.1:2000MAY19 300 848 forward 3Synaptophysin Synaptophysin/synaptoporin 2.10E−20 11LG:352559.1:2000MAY19 125 313 forward 2 KRAB KRAB box 1.60E−41 12LG:247384.1:2000MAY19 182 256 forward 2 zf-C3HC4 Zinc finger, C3HC4 type(RING finger) 1.80E−06 13 LG:403872.1:2000MAY19 717 1187 forward 3 PAP2PAP2 superfamily 1.80E−09 14 LG:1135213.1:2000MAY19 340 531 forward 1T-box T-box 8.80E−27 15 LG:474284.2:2000MAY19 73 195 forward 1 zf-C3HC4Zinc finger, C3HC4 type (RING finger) 1.20E−13 16 LG:342147.1:2000MAY19290 469 forward 2 crystallin Alpha crystallin A chain, N terminal3.10E−09 16 LG:342147.1:2000MAY19 452 628 forward 2 HSP20 Hsp20/alphacrystallin family 7.20E−12 17 LG:1097300.1:2000MAY19 59 250 forward 2rrm RNA recognition motif. (a.k.a. RRM, RBC 4.10E−16 18LG:444850.9:2000MAY19 190 1290 forward 1 GBP Guanylate-binding protein4.20E−247 19 LG:402231.6:2000MAY19 258 380 forward 3 zf-C3HC4 Zincfinger, C3HC4 type (RING finger) 4.30E−05 20 LG:1076157.1:2000MAY19 180320 forward 3 KRAB KRAB box 3.40E−18 21 LG:1083142.1:2000MAY19 129 320forward 3 KRAB KRAB box 2.00E−42 22 LG:1083264.1:2000MAY19 440 628forward 2 KRAB KRAB box 2.30E−33 23 LG:350793.2:2000MAY19 570 722forward 3 Kelch Kelch motif 2.70E−11 24 LG:408751.3:2000MAY19 194 1051forward 2 Dihydrooratase Dihydroorotase-like 5.50E−07 25LI:336120.1:2000MAY01 232 1398 forward 1 Glypican Glypican 9.90E−141 25LI:336120.1:2000MAY01 1476 1907 forward 3 Glypican Glypican 8.60E−70 25LI:336120.1:2000MAY01 503 775 forward 2 Glypican Glypican 3.50E−46 26LI:234104.2:2000MAY01 2517 3002 forward 3 IRK Inward rectifier potassiumchannel 8.70E−111 26 LI:234104.2:2000MAY01 2965 3507 forward 1 IRKInward rectifier potassium channel 9.20E−111 27 LI:450887.1:2000MAY01 48344 forward 3 Ribosomal_L36e Ribosomal protein L36e 6.90E−41 28LI:119992.3:2000MAY01 788 925 forward 2 Kelch Kelch motif 1.50E−09 29LI:197241.2:2000MAY01 1243 1407 forward 1 RCC1 Regulator of chromosomecondensation 1.60E−04 30 LI:406860.20:2000MAY01 228 407 forward 3 igImmunoglobulin domain 1.90E−08 31 LI:142384.1:2000MAY01 318 791 forward3 UQ_con Ubiquitin-conjugating enzyme 1.40E−16 32 LI:895427.1:2000MAY01437 907 forward 2 RhoGAP RhoGAP domain 1.20E−40 33 LI:757439.1:2000MAY011040 1162 forward 2 zf-C3HC4 Zinc finger, C3HC4 type (RING finger)7.20E−10 34 LI:1144066.1:2000MAY01 222 365 forward 3 jmjN jmjN domain2.80E−23 35 LI:243660.4:2000MAY01 316 522 forward 1 HMG_box HMG (highmobility group) box 8.60E−17 36 LI:334386.1:2000MAY01 272 370 forward 2ank Ank repeat 4.90E−08 36 LI:334386.1:2000MAY01 735 833 forward 3 ankAnk repeat 4.50E−05 37 LI:347572.1:2000MAY01 130 1878 forward 1Peptidase_M2 Angiotensin-converting enzyme 2.60E−05 38LI:817314.1:2000MAY01 934 2034 forward 1 Trans_recep Transient receptor6.50E−260 38 LI:817314.1:2000MAY01 1929 2321 forward 3 Trans_recepTransient receptor 2.20E−81 39 LI:000290.1:2000MAY01 960 1040 forward 3zf-CCCH Zinc finger C-x8-C-x5-C-x3-H type (and

7.70E−04 40 LI:023518.3:2000MAY01 195 845 forward 3 vATP- ATP synthase(C/AC39) subunit 5.30E−38 synt_AC39 41 LI:1084246.1:2000MAY01 1443 1733forward 3 cadherin Cadherin domain 2.30E−20 41 LI:1084246.1:2000MAY01875 1150 forward 2 cadherin Cadherin domain 6.60E−17 42LI:1165828.1:2000MAY01 1421 1705 forward 2 cadherin Cadherin domain1.30E−19 43 LI:007302.1:2000MAY01 1646 1810 forward 2 LRRCT Leucine richrepeat C-terminal domain 2.60E−13 43 LI:007302.1:2000MAY01 1991 2455forward 2 TIR TIR domain 3.50E−37 44 LI:236386.4:2000MAY01 677 850forward 2 SH3 SH3 domain 5.20E−07 45 LI:252904.5:2000MAY01 358 495forward 1 Kelch Kelch motif 3.80E−07

TABLE 3 Domain SEQ ID NO: Template ID Start Stop Frame Type Topology 1LG:977683.1:2000FEB18 373 459 forward 1 TM N in 1 LG:977683.1:2000FEB18657 731 forward 3 TM N out 2 LG:893050.1:2000FEB18 15 101 forward 3 TM Nout 3 LG:980153.1:2000FEB18 313 375 forward 1 TM N out 3LG:980153.1:2000FEB18 391 453 forward 1 TM N out 3 LG:980153.1:2000FEB18278 364 forward 2 TM N out 3 LG:980153.1:2000FEB18 416 493 forward 2 TMN out 3 LG:980153.1:2000FEB18 809 871 forward 2 TM N out 3LG:980153.1:2000FEB18 902 964 forward 2 TM N out 3 LG:980153.1:2000FEB181181 1264 forward 2 TM N out 3 LG:980153.1:2000FEB18 1427 1510 forward 2TM N out 3 LG:980153.1:2000FEB18 1733 1798 forward 2 TM N out 3LG:980153.1:2000FEB18 1868 1954 forward 2 TM N out 3LG:980153.1:2000FEB18 2141 2227 forward 2 TM N out 3LG:980153.1:2000FEB18 2261 2308 forward 2 TM N out 3LG:980153.1:2000FEB18 60 125 forward 3 TM N in 3 LG:980153.1:2000FEB18402 476 forward 3 TM N in 3 LG:980153.1:2000FEB18 2031 2081 forward 3 TMN in 3 LG:980153.1:2000FEB18 2142 2213 forward 3 TM N in 5LG:475551.1:2000FEB18 2134 2208 forward 1 TM N in 5LG:475551.1:2000FEB18 2039 2125 forward 2 TM N out 5LG:475551.1:2000FEB18 1167 1217 forward 3 TM N in 6LG:481407.2:2000FEB18 874 927 forward 1 TM 6 LG:481407.2:2000FEB18 9491035 forward 1 TM 6 LG:481407.2:2000FEB18 1081 1161 forward 1 TM 6LG:481407.2:2000FEB18 1510 1584 forward 1 TM 6 LG:481407.2:2000FEB181355 1435 forward 2 TM N out 6 LG:481407.2:2000FEB18 1439 1525 forward 2TM N out 6 LG:481407.2:2000FEB18 1326 1409 forward 3 TM N in 6LG:481407.2:2000FEB18 1446 1526 forward 3 TM N in 6LG:481407.2:2000FEB18 1545 1616 forward 3 TM N in 7LI:443580.1:2000FEB01 488 574 forward 2 TM N out 10LG:171377.1:2000MAY19 318 386 forward 3 TM N in 10 LG:171377.1:2000MAY19549 635 forward 3 TM N in 10 LG:171377.1:2000MAY19 669 740 forward 3 TMN in 12 LG:247384.1:2000MAY19 1381 1461 forward 1 TM N in 12LG:247384.1:2000MAY19 1624 1710 forward 1 TM N in 12LG:247384.1:2000MAY19 1409 1495 forward 2 TM N in 12LG:247384.1:2000MAY19 1395 1481 forward 3 TM N in 12LG:247384.1:2000MAY19 1617 1679 forward 3 TM N in 13LG:403872.1:2000MAY19 535 621 forward 1 TM N in 13 LG:403872.1:2000MAY191360 1446 forward 1 TM N in 13 LG:403872.1:2000MAY19 1522 1581 forward 1TM N in 13 LG:403872.1:2000MAY19 1828 1902 forward 1 TM N in 13LG:403872.1:2000MAY19 1957 2022 forward 1 TM N in 13LG:403872.1:2000MAY19 299 349 forward 2 TM N in 13 LG:403872.1:2000MAY191361 1423 forward 2 TM N in 13 LG:403872.1:2000MAY19 1439 1501 forward 2TM N in 13 LG:403872.1:2000MAY19 1553 1627 forward 2 TM N in 13LG:403872.1:2000MAY19 1859 1918 forward 2 TM N in 13LG:403872.1:2000MAY19 2027 2110 forward 2 TM N in 13LG:403872.1:2000MAY19 2117 2203 forward 2 TM N in 13LG:403872.1:2000MAY19 369 452 forward 3 TM N in 13 LG:403872.1:2000MAY19549 635 forward 3 TM N in 13 LG:403872.1:2000MAY19 708 785 forward 3 TMN in 13 LG:403872.1:2000MAY19 1101 1187 forward 3 TM N in 13LG:403872.1:2000MAY19 1419 1505 forward 3 TM N in 13LG:403872.1:2000MAY19 1575 1661 forward 3 TM N in 13LG:403872.1:2000MAY19 2115 2192 forward 3 TM N in 13LG:403872.1:2000MAY19 2226 2273 forward 3 TM N in 14LG:1135213.1:2000MAY19 41 127 forward 2 TM N out 14LG:1135213.1:2000MAY19 215 274 forward 2 TM N out 14LG:1135213.1:2000MAY19 293 379 forward 2 TM N out 14LG:1135213.1:2000MAY19 389 475 forward 2 TM N out 16LG:342147.1:2000MAY19 142 204 forward 1 TM N out 16LG:342147.1:2000MAY19 171 251 forward 3 TM N out 17LG:1097300.1:2000MAY19 487 564 forward 1 TM 17 LG:1097300.1:2000MAY19805 891 forward 1 TM 17 LG:1097300.1:2000MAY19 1372 1458 forward 1 TM 17LG:1097300.1:2000MAY19 668 754 forward 2 TM N out 17LG:1097300.1:2000MAY19 803 874 forward 2 TM N out 17LG:1097300.1:2000MAY19 1358 1441 forward 2 TM N out 17LG:1097300.1:2000MAY19 522 578 forward 3 TM N in 17LG:1097300.1:2000MAY19 750 836 forward 3 TM N in 17LG:1097300.1:2000MAY19 894 956 forward 3 TM N in 17LG:1097300.1:2000MAY19 1068 1145 forward 3 TM N in 18LG:444850.9:2000MAY19 253 315 forward 1 TM N in 19 LG:402231.6:2000MAY19407 484 forward 2 TM N in 23 LG:350793.2:2000MAY19 148 222 forward 1 TMN in 23 LG:350793.2:2000MAY19 316 384 forward 1 TM N in 23LG:350793.2:2000MAY19 1144 1215 forward 1 TM N in 23LG:350793.2:2000MAY19 1231 1293 forward 1 TM N in 23LG:350793.2:2000MAY19 1339 1425 forward 1 TM N in 23LG:350793.2:2000MAY19 1459 1521 forward 1 TM N in 23LG:350793.2:2000MAY19 1582 1662 forward 1 TM N in 23LG:350793.2:2000MAY19 1882 1953 forward 1 TM N in 23LG:350793.2:2000MAY19 1514 1600 forward 2 TM 23 LG:350793.2:2000MAY192135 2221 forward 2 TM 23 LG:350793.2:2000MAY19 1422 1493 forward 3 TM23 LG:350793.2:2000MAY19 2268 2354 forward 3 TM 24 LG:408751.3:2000MAY191202 1264 forward 2 TM N out 24 LG:408751.3:2000MAY19 1137 1223 forward3 TM N in 25 LI:336120.1:2000MAY01 241 297 forward 1 TM N in 25LI:336120.1:2000MAY01 616 702 forward 1 TM N in 25 LI:336120.1:2000MAY011141 1200 forward 1 TM N in 25 LI:336120.1:2000MAY01 2524 2598 forward 1TM N in 25 LI:336120.1:2000MAY01 1163 1213 forward 2 TM N in 25LI:336120.1:2000MAY01 1922 1972 forward 2 TM N in 25LI:336120.1:2000MAY01 2060 2119 forward 2 TM N in 25LI:336120.1:2000MAY01 2510 2596 forward 2 TM N in 25LI:336120.1:2000MAY01 663 749 forward 3 TM N in 25 LI:336120.1:2000MAY011380 1445 forward 3 TM N in 25 LI:336120.1:2000MAY01 1839 1925 forward 3TM N in 25 LI:336120.1:2000MAY01 2148 2234 forward 3 TM N in 25LI:336120.1:2000MAY01 2418 2471 forward 3 TM N in 25LI:336120.1:2000MAY01 2499 2585 forward 3 TM N in 26LI:234104.2:2000MAY01 1873 1947 forward 1 TM N out 26LI:234104.2:2000MAY01 2155 2241 forward 1 TM N out 26LI:234104.2:2000MAY01 3616 3690 forward 1 TM N out 26LI:234104.2:2000MAY01 1112 1168 forward 2 TM N in 26LI:234104.2:2000MAY01 2216 2302 forward 2 TM N in 26LI:234104.2:2000MAY01 3632 3718 forward 2 TM N in 26LI:234104.2:2000MAY01 3998 4045 forward 2 TM N in 26LI:234104.2:2000MAY01 1314 1400 forward 3 TM N in 26LI:234104.2:2000MAY01 2172 2258 forward 3 TM N in 26LI:234104.2:2000MAY01 2607 2684 forward 3 TM N in 26LI:234104.2:2000MAY01 2739 2798 forward 3 TM N in 26LI:234104.2:2000MAY01 2841 2891 forward 3 TM N in 26LI:234104.2:2000MAY01 3621 3707 forward 3 TM N in 26LI:234104.2:2000MAY01 4080 4145 forward 3 TM N in 28LI:119992.3:2000MAY01 22 102 forward 1 TM N out 28 LI:119992.3:2000MAY01151 237 forward 1 TM N out 28 LI:119992.3:2000MAY01 1444 1530 forward 1TM N out 28 LI:119992.3:2000MAY01 1603 1683 forward 1 TM N out 28LI:119992.3:2000MAY01 1729 1809 forward 1 TM N out 28LI:119992.3:2000MAY01 2197 2253 forward 1 TM N out 28LI:119992.3:2000MAY01 2269 2355 forward 1 TM N out 28LI:119992.3:2000MAY01 2989 3075 forward 1 TM N out 28LI:119992.3:2000MAY01 3163 3249 forward 1 TM N out 28LI:119992.3:2000MAY01 1247 1333 forward 2 TM N in 28LI:119992.3:2000MAY01 1538 1606 forward 2 TM N in 28LI:119992.3:2000MAY01 2207 2293 forward 2 TM N in 28LI:119992.3:2000MAY01 2756 2812 forward 2 TM N in 28LI:119992.3:2000MAY01 3098 3169 forward 2 TM N in 28LI:119992.3:2000MAY01 3281 3343 forward 2 TM N in 28LI:119992.3:2000MAY01 3356 3418 forward 2 TM N in 28LI:119992.3:2000MAY01 120 188 forward 3 TM N in 28 LI:119992.3:2000MAY01627 689 forward 3 TM N in 28 LI:119992.3:2000MAY01 708 770 forward 3 TMN in 28 LI:119992.3:2000MAY01 1425 1511 forward 3 TM N in 28LI:119992.3:2000MAY01 1782 1868 forward 3 TM N in 28LI:119992.3:2000MAY01 2223 2306 forward 3 TM N in 28LI:119992.3:2000MAY01 2757 2843 forward 3 TM N in 28LI:119992.3:2000MAY01 3027 3113 forward 3 TM N in 28LI:119992.3:2000MAY01 3213 3275 forward 3 TM N in 28LI:119992.3:2000MAY01 3312 3374 forward 3 TM N in 29LI:197241.2:2000MAY01 289 369 forward 1 TM N out 29LI:197241.2:2000MAY01 430 507 forward 1 TM N out 29LI:197241.2:2000MAY01 799 861 forward 1 TM N out 29LI:197241.2:2000MAY01 889 951 forward 1 TM N out 29LI:197241.2:2000MAY01 1798 1863 forward 1 TM N out 29LI:197241.2:2000MAY01 1930 2016 forward 1 TM N out 29LI:197241.2:2000MAY01 2101 2148 forward 1 TM N out 29LI:197241.2:2000MAY01 2206 2262 forward 1 TM N out 29LI:197241.2:2000MAY01 416 499 forward 2 TM N out 29LI:197241.2:2000MAY01 812 862 forward 2 TM N out 29LI:197241.2:2000MAY01 1226 1309 forward 2 TM N out 29LI:197241.2:2000MAY01 1475 1558 forward 2 TM N out 29LI:197241.2:2000MAY01 2210 2296 forward 2 TM N out 29LI:197241.2:2000MAY01 60 125 forward 3 TM N in 29 LI:197241.2:2000MAY01333 395 forward 3 TM N in 29 LI:197241.2:2000MAY01 441 503 forward 3 TMN in 29 LI:197241.2:2000MAY01 2223 2300 forward 3 TM N in 31LI:142384.1:2000MAY01 367 432 forward 1 TM N out 31LI:142384.1:2000MAY01 93 155 forward 3 TM N out 32 LI:895427.1:2000MAY011796 1879 forward 2 TM N in 32 LI:895427.1:2000MAY01 1656 1724 forward 3TM N in 33 LI:757439.1:2000MAY01 253 312 forward 1 TM N in 33LI:757439.1:2000MAY01 817 900 forward 1 TM N in 33 LI:757439.1:2000MAY011507 1572 forward 1 TM N in 33 LI:757439.1:2000MAY01 1615 1677 forward 1TM N in 33 LI:757439.1:2000MAY01 1696 1758 forward 1 TM N in 33LI:757439.1:2000MAY01 1834 1899 forward 1 TM N in 33LI:757439.1:2000MAY01 1969 2043 forward 1 TM N in 33LI:757439.1:2000MAY01 2107 2193 forward 1 TM N in 33LI:757439.1:2000MAY01 2506 2586 forward 1 TM N in 33LI:757439.1:2000MAY01 815 901 forward 2 TM N out 33LI:757439.1:2000MAY01 1634 1720 forward 2 TM N out 33LI:757439.1:2000MAY01 1796 1882 forward 2 TM N out 33LI:757439.1:2000MAY01 1952 2026 forward 2 TM N out 33LI:757439.1:2000MAY01 2486 2563 forward 2 TM N out 33LI:757439.1:2000MAY01 783 869 forward 3 TM N in 33 LI:757439.1:2000MAY01996 1049 forward 3 TM N in 33 LI:757439.1:2000MAY01 1545 1631 forward 3TM N in 33 LI:757439.1:2000MAY01 2115 2174 forward 3 TM N in 35LI:243660.4:2000MAY01 1247 1333 forward 2 TM N in 36LI:334386.1:2000MAY01 538 621 forward 1 TM 36 LI:334386.1:2000MAY01 9221008 forward 1 TM 36 LI:334386.1:2000MAY01 1087 1173 forward 1 TM 36LI:334386.1:2000MAY01 1468 1530 forward 1 TM 36 LI:334386.1:2000MAY011570 1632 forward 1 TM 36 LI:334386.1:2000MAY01 2731 2802 forward 1 TM36 LI:334386.1:2000MAY01 2992 3054 forward 1 TM 36 LI:334386.1:2000MAY013325 3387 forward 1 TM 36 LI:334386.1:2000MAY01 3406 3468 forward 1 TM36 LI:334386.1:2000MAY01 3487 3570 forward 1 TM 36 LI:334386.1:2000MAY013766 3852 forward 1 TM 36 LI:334386.1:2000MAY01 4006 4077 forward 1 TM36 LI:334386.1:2000MAY01 4342 4416 forward 1 TM 36 LI:334386.1:2000MAY014615 4686 forward 1 TM 36 LI:334386.1:2000MAY01 4747 4833 forward 1 TM36 LI:334386.1:2000MAY01 5062 5124 forward 1 TM 36 LI:334386.1:2000MAY015140 5202 forward 1 TM 36 LI:334386.1:2000MAY01 5227 5289 forward 1 TM36 LI:334386.1:2000MAY01 5563 5649 forward 1 TM 36 LI:334386.1:2000MAY011235 1321 forward 2 TM N in 36 LI:334386.1:2000MAY01 2423 2476 forward 2TM N in 36 LI:334386.1:2000MAY01 2702 2764 forward 2 TM N in 36LI:334386.1:2000MAY01 2792 2854 forward 2 TM N in 36LI:334386.1:2000MAY01 3086 3172 forward 2 TM N in 36LI:334386.1:2000MAY01 3302 3355 forward 2 TM N in 36LI:334386.1:2000MAY01 3452 3517 forward 2 TM N in 36LI:334386.1:2000MAY01 3920 4006 forward 2 TM N in 36LI:334386.1:2000MAY01 4064 4144 forward 2 TM N in 36LI:334386.1:2000MAY01 4250 4318 forward 2 TM N in 36LI:334386.1:2000MAY01 4331 4402 forward 2 TM N in 36LI:334386.1:2000MAY01 4523 4576 forward 2 TM N in 36LI:334386.1:2000MAY01 4586 4669 forward 2 TM N in 36LI:334386.1:2000MAY01 4772 4855 forward 2 TM N in 36LI:334386.1:2000MAY01 5039 5125 forward 2 TM N in 36LI:334386.1:2000MAY01 5498 5584 forward 2 TM N in 36LI:334386.1:2000MAY01 30 116 forward 3 TM N in 36 LI:334386.1:2000MAY01324 380 forward 3 TM N in 36 LI:334386.1:2000MAY01 387 470 forward 3 TMN in 36 LI:334386.1:2000MAY01 531 608 forward 3 TM N in 36LI:334386.1:2000MAY01 1362 1448 forward 3 TM N in 36LI:334386.1:2000MAY01 1539 1625 forward 3 TM N in 36LI:334386.1:2000MAY01 2232 2279 forward 3 TM N in 36LI:334386.1:2000MAY01 2580 2651 forward 3 TM N in 36LI:334386.1:2000MAY01 2757 2822 forward 3 TM N in 36LI:334386.1:2000MAY01 2820 2870 forward 3 TM N in 36LI:334386.1:2000MAY01 3282 3368 forward 3 TM N in 36LI:334386.1:2000MAY01 3510 3596 forward 3 TM N in 36LI:334386.1:2000MAY01 3981 4064 forward 3 TM N in 36LI:334386.1:2000MAY01 4356 4427 forward 3 TM N in 36LI:334386.1:2000MAY01 4464 4544 forward 3 TM N in 36LI:334386.1:2000MAY01 4959 5024 forward 3 TM N in 36LI:334386.1:2000MAY01 5601 5687 forward 3 TM N in 37LI:347572.1:2000MAY01 790 876 forward 1 TM N in 37 LI:347572.1:2000MAY011354 1434 forward 1 TM N in 37 LI:347572.1:2000MAY01 2425 2511 forward 1TM N in 37 LI:347572.1:2000MAY01 2599 2685 forward 1 TM N in 37LI:347572.1:2000MAY01 2686 2757 forward 1 TM N in 37LI:347572.1:2000MAY01 3133 3207 forward 1 TM N in 37LI:347572.1:2000MAY01 1184 1255 forward 2 TM 37 LI:347572.1:2000MAY012264 2350 forward 2 TM 37 LI:347572.1:2000MAY01 2597 2665 forward 2 TM37 LI:347572.1:2000MAY01 2942 3028 forward 2 TM 37 LI:347572.1:2000MAY013137 3199 forward 2 TM 37 LI:347572.1:2000MAY01 3227 3289 forward 2 TM37 LI:347572.1:2000MAY01 129 215 forward 3 TM N in 37LI:347572.1:2000MAY01 969 1046 forward 3 TM N in 37LI:347572.1:2000MAY01 1947 2033 forward 3 TM N in 37LI:347572.1:2000MAY01 2208 2288 forward 3 TM N in 37LI:347572.1:2000MAY01 2412 2477 forward 3 TM N in 37LI:347572.1:2000MAY01 2604 2684 forward 3 TM N in 37LI:347572.1:2000MAY01 2739 2795 forward 3 TM N in 38LI:817314.1:2000MAY01 460 546 forward 1 TM 38 LI:817314.1:2000MAY01 11921278 forward 1 TM 38 LI:817314.1:2000MAY01 1318 1386 forward 1 TM 38LI:817314.1:2000MAY01 1423 1485 forward 1 TM 38 LI:817314.1:2000MAY011537 1599 forward 1 TM 38 LI:817314.1:2000MAY01 1630 1692 forward 1 TM38 LI:817314.1:2000MAY01 1756 1842 forward 1 TM 38 LI:817314.1:2000MAY011930 1992 forward 1 TM 38 LI:817314.1:2000MAY01 2032 2094 forward 1 TM38 LI:817314.1:2000MAY01 2860 2946 forward 1 TM 38 LI:817314.1:2000MAY013127 3213 forward 1 TM 38 LI:817314.1:2000MAY01 362 448 forward 2 TM Nin 38 LI:817314.1:2000MAY01 3158 3244 forward 2 TM N in 38LI:817314.1:2000MAY01 30 95 forward 3 TM N out 38 LI:817314.1:2000MAY011239 1301 forward 3 TM N out 38 LI:817314.1:2000MAY01 1785 1865 forward3 TM N out 38 LI:817314.1:2000MAY01 1920 2000 forward 3 TM N out 38LI:817314.1:2000MAY01 3189 3269 forward 3 TM N out 39LI:000290.1:2000MAY01 1003 1065 forward 1 TM N in 39LI:000290.1:2000MAY01 1075 1137 forward 1 TM N in 39LI:000290.1:2000MAY01 1195 1248 forward 1 TM N in 39LI:000290.1:2000MAY01 767 844 forward 2 TM 39 LI:000290.1:2000MAY01 882932 forward 3 TM N in 40 LI:023518.3:2000MAY01 28 108 forward 1 TM N out40 LI:023518.3:2000MAY01 20 106 forward 2 TM N in 41LI:1084246.1:2000MAY01 178 264 forward 1 TM N out 41LI:1084246.1:2000MAY01 2686 2760 forward 1 TM N out 41LI:1084246.1:2000MAY01 2932 3003 forward 1 TM N out 41LI:1084246.1:2000MAY01 3097 3159 forward 1 TM N out 41LI:1084246.1:2000MAY01 3184 3246 forward 1 TM N out 41LI:1084246.1:2000MAY01 3352 3405 forward 1 TM N out 41LI:1084246.1:2000MAY01 3409 3480 forward 1 TM N out 41LI:1084246.1:2000MAY01 3526 3609 forward 1 TM N out 41LI:1084246.1:2000MAY01 200 253 forward 2 TM N in 41LI:1084246.1:2000MAY01 2171 2254 forward 2 TM N in 41LI:1084246.1:2000MAY01 2654 2734 forward 2 TM N in 41LI:1084246.1:2000MAY01 3065 3142 forward 2 TM N in 41LI:1084246.1:2000MAY01 3284 3358 forward 2 TM N in 41LI:1084246.1:2000MAY01 3479 3553 forward 2 TM N in 41LI:1084246.1:2000MAY01 582 641 forward 3 TM N out 41LI:1084246.1:2000MAY01 2127 2213 forward 3 TM N out 41LI:1084246.1:2000MAY01 2457 2543 forward 3 TM N out 41LI:1084246.1:2000MAY01 2580 2666 forward 3 TM N out 41LI:1084246.1:2000MAY01 2751 2813 forward 3 TM N out 41LI:1084246.1:2000MAY01 2826 2888 forward 3 TM N out 41LI:1084246.1:2000MAY01 2961 3047 forward 3 TM N out 41LI:1084246.1:2000MAY01 3249 3335 forward 3 TM N out 41LI:1084246.1:2000MAY01 3429 3515 forward 3 TM N out 42LI:1165828.1:2000MAY01 61 147 forward 1 TM N out 42LI:1165828.1:2000MAY01 244 312 forward 1 TM N out 42LI:1165828.1:2000MAY01 454 510 forward 1 TM N out 42LI:1165828.1:2000MAY01 3664 3750 forward 1 TM N out 42LI:1165828.1:2000MAY01 3937 4023 forward 1 TM N out 42LI:1165828.1:2000MAY01 4600 4653 forward 1 TM N out 42LI:1165828.1:2000MAY01 4855 4941 forward 1 TM N out 42LI:1165828.1:2000MAY01 5047 5133 forward 1 TM N out 42LI:1165828.1:2000MAY01 5227 5298 forward 1 TM N out 42LI:1165828.1:2000MAY01 5311 5388 forward 1 TM N out 42LI:1165828.1:2000MAY01 5491 5577 forward 1 TM N out 42LI:1165828.1:2000MAY01 5800 5871 forward 1 TM N out 42LI:1165828.1:2000MAY01 227 301 forward 2 TM N in 42LI:1165828.1:2000MAY01 713 775 forward 2 TM N in 42LI:1165828.1:2000MAY01 1769 1819 forward 2 TM N in 42LI:1165828.1:2000MAY01 2759 2845 forward 2 TM N in 42LI:1165828.1:2000MAY01 3869 3928 forward 2 TM N in 42LI:1165828.1:2000MAY01 4688 4774 forward 2 TM N in 42LI:1165828.1:2000MAY01 5048 5116 forward 2 TM N in 42LI:1165828.1:2000MAY01 5531 5617 forward 2 TM N in 42LI:1165828.1:2000MAY01 5816 5893 forward 2 TM N in 42LI:1165828.1:2000MAY01 39 113 forward 3 TM N out 42LI:1165828.1:2000MAY01 906 968 forward 3 TM N out 42LI:1165828.1:2000MAY01 1602 1688 forward 3 TM N out 42LI:1165828.1:2000MAY01 3471 3557 forward 3 TM N out 42LI:1165828.1:2000MAY01 3558 3608 forward 3 TM N out 42LI:1165828.1:2000MAY01 4203 4289 forward 3 TM N out 42LI:1165828.1:2000MAY01 4749 4835 forward 3 TM N out 42LI:1165828.1:2000MAY01 5625 5690 forward 3 TM N out 42LI:1165828.1:2000MAY01 5847 5918 forward 3 TM N out 43LI:007302.1:2000MAY01 346 426 forward 1 TM N in 43 LI:007302.1:2000MAY012638 2721 forward 1 TM N in 43 LI:007302.1:2000MAY01 59 145 forward 2 TMN out 43 LI:007302.1:2000MAY01 653 718 forward 2 TM N out 43LI:007302.1:2000MAY01 1799 1885 forward 2 TM N out 43LI:007302.1:2000MAY01 321 407 forward 3 TM N in 43 LI:007302.1:2000MAY01480 566 forward 3 TM N in 43 LI:007302.1:2000MAY01 645 704 forward 3 TMN in 43 LI:007302.1:2000MAY01 807 890 forward 3 TM N in 43LI:007302.1:2000MAY01 1161 1223 forward 3 TM N in 43LI:007302.1:2000MAY01 1236 1298 forward 3 TM N in 43LI:007302.1:2000MAY01 1362 1448 forward 3 TM N in 43LI:007302.1:2000MAY01 1809 1868 forward 3 TM N in 43LI:007302.1:2000MAY01 1998 2084 forward 3 TM N in 43LI:007302.1:2000MAY01 2184 2234 forward 3 TM N in 43LI:007302.1:2000MAY01 2457 2540 forward 3 TM N in 43LI:007302.1:2000MAY01 2595 2681 forward 3 TM N in 44LI:236386.4:2000MAY01 3739 3792 forward 1 TM N out 44LI:236386.4:2000MAY01 53 118 forward 2 TM N out 44 LI:236386.4:2000MAY01218 304 forward 2 TM N out 44 LI:236386.4:2000MAY01 3755 3823 forward 2TM N out 44 LI:236386.4:2000MAY01 2376 2435 forward 3 TM N out 45LI:252904.5:2000MAY01 494 550 forward 2 TM N out 45LI:252904.5:2000MAY01 300 374 forward 3 TM N out

TABLE 4 SEQ ID Component NO: ID Start Stop 1 g5813583 610 959 16817504J1 1 621 1 g1989978 3 264 1 4292280H1 10 242 1 483000R6 11 337 1483000H1 11 252 1 g1424329 14 316 1 3255214H1 107 349 1 1450061H1 131371 1 5388816H1 152 419 1 955673H1 181 406 1 2109273H1 286 547 15980116H1 373 651 1 g828864 376 596 1 3072657H1 380 488 1 2949928H1 416680 1 6016294H1 580 677 1 g1855323 611 695 1 g1623907 611 667 1 g1855498611 933 1 g1751162 689 928 1 1309114T6 716 955 1 1309114F6 716 979 11309114H1 716 971 1 3637614H1 807 1053 1 7065033H1 899 1165 1 6817504H1971 1358 1 6013754H1 978 1245 1 g573231 1034 1316 1 g709283 1034 1322 1g767017 1035 1345 1 g692230 1061 1388 1 1617090H1 1084 1209 1 1617090F61084 1380 1 g1157664 1112 1412 2 6131346H1 1 193 2 6871387H1 125 662 2g2279352 352 634 3 7039759H1 1390 1914 3 6481201H1 1428 1542 3 6929893H11460 1891 3 160750H1 1643 1734 3 6201684H1 1659 2172 3 492554H1 36 275 36710369H1 84 594 3 g770845 369 639 3 6710369J1 538 1037 3 6866894H1 7491339 3 2045879F6 796 1123 3 2045879H1 796 1064 3 g677645 854 1153 3g570913 854 1235 3 2837088H1 1 79 3 g878213 855 1194 3 3637810H1 9051188 3 382301R6 11 244 3 3637810F8 906 1347 3 5516287H1 938 1192 3382301H1 11 273 3 310657H1 983 1184 3 381716R1 11 471 3 054856H1 10271268 3 2676843H1 1102 1294 3 2865460H1 1182 1413 3 5983503H1 1223 1521 33296833H1 24 289 3 492559R1 36 564 3 3903656H1 1288 1501 3 2554026H11322 1591 3 g1894266 1326 1800 3 3151953H1 2028 2266 3 6357422H1 20562344 3 382301T6 2063 2619 3 2498615F6 2077 2500 3 2498615H1 2077 2310 3492559F1 2104 2658 3 2684917H1 1709 1950 3 3898190H1 1917 2210 3381716F1 2106 2658 3 5952437H1 1960 2247 3 4701147H1 2134 2402 3g5435909 2213 2663 3 7067611H1 2254 2764 3 g2563607 2282 2658 31889064H1 2300 2577 3 2400488H1 2302 2549 3 g817549 2307 2667 3 g5669652343 2658 3 g1894154 2354 2658 3 g869609 2394 2667 3 g4291206 2396 27663 g646309 2398 2658 3 3249908H1 2467 2760 3 672907H1 2516 2658 3672763R6 2516 2658 3 672763H1 2516 2658 3 672696H1 2516 2658 3 672763T62516 2621 4 g1939101 219 609 4 1749048T6 1 388 5 996489H1 1 289 5996489R6 1 321 5 6807726H1 9 414 5 g1208184 74 603 5 g1146490 110 406 51391557H1 145 273 5 2054016H1 155 406 5 3564377H1 213 498 5 1389469H1365 607 5 6178475H1 288 554 5 2490333H1 461 684 5 1498011F6 497 816 51498011H1 497 735 5 154577H1 512 727 5 2439861H1 600 846 5 6974170H1 6551206 5 5557446H1 723 990 5 6821354J1 725 1336 5 3801324H1 751 1035 5159257H1 753 952 5 1562163H1 801 1030 5 7161127H1 827 1358 5 1840238H1834 989 5 1892815H1 944 1194 5 1893046H1 944 1185 5 1391452H1 962 1131 51391452F6 962 1223 5 1680496H1 1117 1345 5 2132470R6 1120 1456 51265470H1 1149 1401 5 6804038H1 1164 1555 5 3430883H1 1183 1428 52132470H1 1188 1456 5 1515410H1 1224 1442 5 g2056082 1221 1509 5566614H1 1269 1530 5 4780315H1 1290 1553 5 1637781H1 1302 1454 51638827H1 1302 1455 5 1633937H1 1762 1969 5 6821354H1 1419 1971 51390745H1 1433 1557 5 1932110H1 1712 1868 5 1932110F6 1713 1960 51850028H1 1728 1970 5 386578H1 1753 2029 5 1862471H1 1759 1870 54588296H1 1799 1890 5 2028756H1 1816 1890 5 1988349T6 1824 2253 51498011T6 1829 2254 5 6157225H1 1842 2101 5 521110H1 1850 1975 56157733H1 1854 2051 5 4829815H1 1889 1962 5 4411517H1 1907 2157 5541981H1 1927 2155 5 4558860H1 1944 2106 5 1391452T6 1958 2260 52752758H1 1963 2239 5 1807380T6 1965 2250 5 1807042F6 1970 2290 51807042H1 1970 2255 5 2311115H1 1992 2237 5 996489T6 1994 2332 56125387H1 2007 2356 5 4905520H1 2022 2280 5 4671595H1 2027 2277 5318659H1 2041 2291 5 4902185H1 2096 2297 5 g2055975 2105 2298 51219763H1 2110 2288 5 1219763R6 2110 2290 5 1219763T6 2110 2251 51219763T1 2110 2250 5 581809H1 2110 2369 5 g2788727 2119 2369 52753294H1 2255 2364 6 2055577R6 766 1137 6 2055577T6 766 1096 6 g1578280767 1137 6 g4897043 769 1147 6 g1897641 769 1137 6 g3004281 774 1138 66361438H2 776 1335 6 1273945F1 790 1131 6 1273945H1 790 948 6 2558966H1791 1058 6 g2178992 831 1147 6 g1891843 842 1143 6 g1203333 844 1159 6g1141073 845 1135 6 g1728655 851 1143 6 4618322H1 860 1133 6 g3179203882 1147 6 4164817H1 9 261 6 5851107H1 12 270 6 4938618H1 1 285 62096384H1 13 274 6 4938518H1 1 184 6 6133436H1 6 304 6 5218795H1 14 2826 3038155H1 6 294 6 3088308H1 14 285 6 6821608H1 14 578 6 5855412H1 14297 6 2532161H1 6 258 6 5999068H1 6 559 6 g5431297 7 324 6 2715577H1 14256 6 3717266H1 6 312 6 3088671H1 14 251 6 1690850T6 16 558 6 4978332H119 305 6 2525160H1 368 619 6 2811816H1 382 591 6 5285481H1 381 530 6g1923667 380 575 6 2724519H1 385 586 6 4403213H1 397 537 6 2525196H1 368597 6 g2111237 370 592 6 g1155753 370 731 6 g2111348 371 598 6 g3798474371 588 6 g2968466 372 670 6 g1874430 374 675 6 g3933996 376 589 6g2567131 409 663 6 g1422584 429 556 6 g2157052 435 744 6 3092788H1 437722 6 1650634F6 441 871 6 1831391H1 637 867 6 2173245H1 652 888 6768284H1 670 900 6 g2567185 671 1075 6 2522538H1 672 909 6 g3446544 6761136 6 4377572H1 680 948 6 g4242762 685 1135 6 g5444329 685 1147 6g4394905 687 1135 6 g4891466 689 1136 6 4534880T1 604 1111 6 g1422487626 919 6 3213475H1 692 929 6 g3674532 698 1150 6 g3665343 700 1135 6g5365390 705 1135 6 3362353H1 708 848 6 g3737258 707 1140 6 3801387H1711 869 6 g1277444 717 1135 6 6045963H1 722 1176 6 g2236500 716 1139 64024228H1 722 1008 6 g4088002 718 1149 6 3553263H1 754 969 6 g2229274762 1153 6 2055577H1 766 1031 6 5116334H1 19 290 6 1546662H1 19 218 62275605H1 19 291 6 5968841H1 19 591 6 1902261H1 1 288 6 6728620H1 29 5906 1690850F6 29 482 6 1690850H1 29 237 6 5346772H1 29 227 6 5346890H1 29141 6 4151612H1 31 258 6 g2229063 27 371 6 3074071H1 31 308 6 3717427H132 401 6 2467222H1 32 258 6 5687205H1 33 296 6 g2027890 31 188 62864630H1 34 341 6 3837823H1 35 321 6 5978027H1 35 298 6 3841249H1 35236 6 5780416H1 37 313 6 4525495H1 38 294 6 2943180H1 35 281 6 3159688H136 136 6 g2156554 35 459 6 5989823H1 38 334 6 4525695H1 38 287 6774424H1 38 269 6 4376239H1 38 242 6 222536R1 19 533 6 4951501H2 19 3256 5986222H1 21 289 6 4782312H1 19 258 6 222536H1 19 150 6 6152094H1 26301 6 3365655H1 27 286 6 2098005H1 27 209 6 2874828H1 27 311 6 4748012H129 297 6 5122477H1 27 278 6 5516387H1 27 270 6 5695974H1 27 203 64994832H1 36 185 6 g1728758 40 325 6 5993725H1 40 342 6 5995510H1 40 3306 g4329715 40 406 6 2894305H1 47 310 6 2719394T6 303 625 6 g5658221 327736 6 5857676H1 296 564 6 5726056H2 297 676 6 2097760H1 300 546 62873090H1 329 605 6 3136434H1 334 597 6 g1646811 339 596 6 2738075F6 321767 6 2738075H1 321 564 6 2719394F6 318 683 6 2719394H1 267 521 6g5527461 339 586 6 g2437242 340 551 6 4724150H1 343 607 6 g1312816 346778 6 4787470H1 360 597 6 5003922H1 362 616 6 6156796H1 87 345 62895320H1 43 273 6 4665825H1 96 339 6 3232485H1 44 316 6 2399837H1 98322 6 6904948H1 101 462 6 6411519H1 45 554 6 035304H1 55 324 6 4573015H1116 388 6 5609131H1 123 365 6 g3598018 135 590 6 g3432506 136 593 6g5431490 144 323 6 g1646810 57 324 6 g2555607 156 500 6 g1578371 53 1986 g2229126 158 593 6 g3229125 173 598 6 g3898868 173 593 6 g4452177 180323 6 g3182012 205 593 6 790141R1 222 746 6 790141H1 222 456 6 3599189H1229 519 6 g2204943 229 593 6 3258218H1 232 529 6 g2355330 244 592 6g2882852 65 382 6 g1950563 70 330 6 1548020H1 72 301 6 2823270H1 250 5386 2873603H1 257 537 6 2755517H1 79 346 6 3718262H1 81 391 6 915491R6 260597 6 915491H1 260 569 6 4979613H1 276 550 6 6821608J1 278 791 63246153H1 278 516 6 4008733H1 281 559 6 4989076H1 497 752 6 g5850851 503739 6 g4738819 504 739 6 g5849856 504 739 6 6365612H1 519 816 65183801H1 525 789 6 3706413H1 529 812 6 4828553H1 532 762 6 2604912H1539 791 6 g2107086 553 977 6 g5769539 555 733 6 5576107H1 559 800 6g1891969 565 972 6 3620132H1 31 324 6 4605074H1 598 846 6 1650642F6 441832 6 3443641H1 484 742 6 g3889543 490 917 6 g3095491 492 586 62738075T6 494 1096 6 4534880H1 441 701 6 4277322H1 497 751 6 4989476F8496 967 6 1650634H1 441 687 6 g2575167 443 843 6 3718361H1 456 769 63267371H1 457 700 6 1902161H1 462 586 6 5056004H1 465 746 6 g3751871 477736 6 2997314H1 482 786 6 2996840H1 483 745 6 4276994H1 497 635 6g1923480 981 1130 6 6550669H1 1020 1619 6 g4083790 1388 1829 6 4700302H11388 1666 6 g3770915 1402 1832 6 g1224283 1032 1442 6 g2767747 1055 11356 2539090H1 1087 1334 6 1773532H1 1179 1391 6 6045963J1 1211 1801 61650634T6 1270 1789 6 g4373516 1308 1756 7 g2524924 315 730 7 g2161228313 724 7 g3802198 329 703 7 g3147794 231 688 7 g2162211 119 550 72497157H1 78 310 7 2854513H1 1 290 8 1985316H1 1 269 8 1985316R6 1 310 8197972T6 43 445 8 197972H1 43 274 8 197972R6 43 457 9 7197754H2 1 582 10g5810426 1 449 10 g2219401 2 423 10 g4329377 27 489 10 g2537784 172 66910 g1376965 259 669 10 4983705H1 270 539 10 7269840H1 339 848 116453567H1 1 503 11 4052122H1 185 457 11 4052122F7 185 636 11 g3897399255 371 12 973628H1 996 1226 12 3014231H1 1097 1369 12 975169T6 11121714 12 3042767T6 1122 1713 12 6218188H1 1165 1678 12 5151940H1 12161440 12 975304T6 1231 1709 12 5531975T6 1266 1741 12 3577265H1 1286 159812 3016255H1 1291 1599 12 970343R6 1304 1757 12 970343H1 1304 1606 12970343T6 1322 1714 12 3575519H1 1334 1616 12 5153116H1 1345 1469 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2201 362760124R6 1934 2378 36 g6330616 228 5624 36 2733278F6 745 1284 363994147H1 5353 5628 36 6883937J1 1 549 37 70554791V1 269 836 3770555906V1 482 1070 37 70557145V1 488 1152 37 70328701D1 115 602 3770557446V1 1746 2364 37 70557024V1 1777 2435 37 70326732D1 1800 2134 3770326508D1 1800 1870 37 71304277V1 1830 2463 37 71156493V1 1852 2469 3771303442V1 1864 2504 37 5542815H1 1873 2025 37 71157532V1 1881 2356 3770555668V1 1893 2524 37 70555958V1 1930 2595 37 70555146V1 1931 2563 3771303538V1 1959 2455 37 71304228V1 1958 2586 37 6496937H1 1967 2501 37305090R6 1971 2342 37 305090H1 1970 2306 37 4598818H1 1996 2251 376349213H2 2054 2378 37 70556404V1 1493 2023 37 3696047F6 1521 2066 373696047H1 1522 1818 37 71158742V1 1536 2128 37 71156538V1 1542 2034 3770327564D1 1550 2005 37 4670450H1 1563 1762 37 71157870V1 1598 2195 3770556820V1 1615 2235 37 6416418H1 1667 1887 37 6389818H1 1667 1987 374518860H1 1672 1933 37 70554892V1 1703 2343 37 70554965V1 1703 2332 376830659J1 1705 2343 37 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4971 5424 42 71188351V1 3626 4086 42 70838919V1 3631 4136 4271188254V1 3638 4239 42 71189595V1 3651 3907 42 70870573V1 3655 4351 4270868067V1 3679 4334 42 70867164V1 3682 4354 42 71230406V1 3685 4227 4270869964V1 3682 4340 42 1817860F6 3725 4287 42 1817860H1 3725 4029 427050051H1 3739 4283 42 70816308V1 4604 5347 42 70813062V1 4615 5238 427103719H1 4627 5050 42 g883091 4613 5038 42 1963922R6 4615 5216 4270825247V1 4615 5083 42 70815988V1 4615 5030 42 70649447V1 4615 5280 4270814603V1 4615 5185 42 70812386V1 4615 5163 42 70813116V1 4615 5137 4270812591V1 4615 5112 42 1963922H1 4615 4860 42 70817149V1 4615 5238 4271190973V1 3394 4015 42 70866857V1 3421 4053 42 70869712V1 3422 4110 4271190024V1 3462 4134 42 71222510V1 3809 4002 42 71229550V1 3828 4582 427317184H2 3840 4515 42 71191575V1 3866 4388 42 71190157V1 3909 4588 4271230422V1 3911 4602 42 70868868V1 3926 4435 42 71191209V1 3938 4502 4271229173V1 3944 4466 42 71191826V1 3939 4349 42 71188071V1 3982 4494 4271222526V1 3993 4351 42 70868437V1 4000 4529 42 70867683V1 4003 4658 4271190956V1 4017 4607 42 70867083V1 4019 4527 42 70869984V1 4019 4488 42g775853 4047 4392 42 71189002V1 4049 4491 42 70870114V1 4057 4751 421963922T6 5617 6180 42 745052H1 5643 5869 42 3333795T6 5681 6181 424421884H1 5703 5956 42 g4989315 5743 6225 42 g3446159 5744 6227 42g5853840 5747 6219 42 2280040T6 5748 6175 42 g4264936 5749 6222 42g5590548 5767 6219 42 2280040R6 5769 6222 42 2280040H1 5769 6044 42g4114692 5775 6229 42 2157793H1 5776 6020 42 g4269881 5783 6222 42g314938 5790 6222 42 5014904T6 5789 6175 42 g1516807 5846 6222 4271190271V1 3599 4339 42 5515021R7 3622 4216 42 71229150V1 3622 4275 4270867419V1 3623 4261 42 g671390 5960 6219 42 g820781 5971 6244 42g668623 6031 6222 42 71221653V1 6103 6222 42 g882914 6021 6129 4271188120V1 4750 4951 42 1267718F1 4756 5198 42 71190911V1 4733 5379 4271188586V1 4756 5397 42 70869357V1 4982 5696 42 g3756453 4981 5424 424776237H1 4985 5261 42 71190506V1 5033 5514 42 6608393T1 5498 6138 425907377H1 5524 5800 42 70870592V1 5528 6173 42 70813957V1 5544 6036 423333795F6 5552 6027 42 3333795H1 5552 5840 42 71188885V1 4599 5206 42g1525426 5842 6222 42 g882983 5853 6245 42 g797506 5865 6230 42 g5871845880 6222 42 70870719V1 5924 6239 42 g814957 5894 6223 42 g822523 59646230 42 g612999 4719 5074 43 g2034169 2102 2394 43 5540505T7 2291 287043 6377332H1 2417 2702 43 4947810H1 2612 2733 43 g5006247 1 2762 435540505F6 953 1415 43 5540505H1 953 1146 43 g2875734 2835 2940 43g3735348 2634 2945 43 5118201T6 2631 2910 43 2749265F6 2448 2923 432749265H1 2448 2714 43 2749265T6 2551 2897 43 537065H1 2429 2663 441452312F1 3288 3835 44 70007188D1 3260 3637 44 g898311 3282 3460 441452312F6 3288 3736 44 1452312H1 3288 3560 44 2599007H1 3312 3589 446325947H1 3442 3749 44 840648H1 3415 3672 44 70012088D1 3420 3797 445852153H1 3426 3701 44 70604010V1 1419 2043 44 6952285H1 1480 2049 444458494F6 1493 1942 44 70608095V1 1492 1936 44 4458494H1 1494 1730 447255931H2 1571 1752 44 6909665J1 1608 2154 44 6969377U1 1616 2026 442272356R6 1622 1941 44 2272356H1 1622 1890 44 70608114V1 1801 1904 446553230H1 1811 2165 44 6559394H1 1811 2428 44 3382113H1 1881 2090 4470606021V1 1880 2259 44 70879980V1 2089 2579 44 2661806F6 2089 2531 442661806H1 2089 2361 44 70879113V1 2089 2545 44 g6476309 2149 2506 442627073H1 2160 2391 44 2627315H1 2160 2389 44 3901711H1 2247 2491 4470887530V1 2263 2344 44 6969302U1 2280 2623 44 70881572V1 2297 2821 445763849H1 2351 2873 44 7256511H1 2398 2905 44 70882796V1 2405 3030 4470886211V1 2434 2594 44 70882791V1 2477 2906 44 70882271V1 2478 2974 4470881365V1 2478 2973 44 70003939D1 2481 2947 44 70012299D1 2481 2829 4470004016D1 2481 3025 44 3572311F6 2487 3077 44 3572311H1 2487 2699 4470005627D1 2487 2687 44 70010847D1 2517 2952 44 7336064H1 2527 2982 4470880257V1 2544 3145 44 70011933D1 2553 3044 44 2272356T6 2566 3001 4470888761V1 2568 2873 44 3011048H1 3342 3641 44 4562117H1 3350 3613 444563263H1 3352 3636 44 70603379V1 1131 1723 44 70603933V1 1153 1782 4470607414V1 1277 1412 44 70607363V1 1042 1396 44 2414751H1 3218 3489 44389997H1 3676 3915 44 6357624H1 3682 3922 44 g3961665 3684 3920 44g6477150 3686 3925 44 1689958F6 3693 3923 44 1689958H1 3693 3907 441689958T6 3698 3880 44 1702166T6 3718 3866 44 3572311T6 3740 3872 44g4649451 3791 3915 44 4099042H2 3816 3927 44 4099042F8 3816 4438 441243554H1 3816 3923 44 g4325490 3834 3915 44 2968601H1 3954 4247 44g5810032 3494 3926 44 7255223H1 3518 3915 44 g2237335 3527 3920 442878117H1 3530 3815 44 g1400734 3536 3915 44 5104505H1 3540 3772 44g4081742 3542 3923 44 1452312T6 3546 3876 44 g898312 3565 3918 446499719H1 3564 3909 44 g4081564 3565 3923 44 g2335900 3599 3920 44g6451467 3602 3915 44 g1521304 3605 3931 44 g4534027 3606 3923 445790863H1 3609 3903 44 5789451H1 3609 3898 44 5787849H1 3609 3915 44g5528373 3621 3920 44 g1516463 3624 3931 44 g5912966 3660 3920 44344685H1 3673 3922 44 2623608H1 3367 3604 44 840648R1 3415 3915 444333836H1 3415 3703 44 70881547V1 3400 3921 44 70886619V1 3404 3634 442414749F6 3218 3747 44 70605048V1 1033 1331 44 7267489H1 1034 1578 446346421H1 3442 3736 44 6317150H1 3442 3746 44 4897563H1 3129 3422 445379052H1 3137 3362 44 3406784H1 3145 3410 44 70008878D1 3156 3637 4470608052V1 1080 1187 44 g3888759 1108 1488 44 2857322H1 2904 3183 4470881851V1 2904 3275 44 792748R1 2910 3533 44 792748H1 2909 3154 44793130H1 2910 3134 44 7159471H1 2922 3506 44 70880131V1 2923 3534 441541872H1 2940 3161 44 684595H1 2941 3207 44 70886274V1 2982 3197 4470886318V1 2982 3196 44 6722223H1 3013 3202 44 2806050H1 3019 3347 441702166F6 3044 3568 44 1702166H1 3044 3271 44 4980587H1 3057 3327 446909665H1 3076 3619 44 4372755H1 3078 3384 44 6074761H1 3079 3396 44685902H1 2605 2829 44 70880726V1 2616 3181 44 2615527H1 2623 2881 4470879436V1 2671 3129 44 70882269V1 2673 3180 44 70887568V1 2676 2818 4470882659V1 2688 3179 44 1438876F1 2686 3071 44 1438880H1 2686 2970 441438876H1 2686 2968 44 2258046H1 2717 2963 44 70003496D1 2721 3284 4470011398D1 2733 3192 44 70882502V1 2739 3418 44 70879669V1 2748 3253 4470006402D1 2745 3309 44 70004115D1 2745 3108 44 70011055D1 2745 3198 4470882244V1 2768 3039 44 70007592D1 2769 2981 44 6479471H1 2787 3356 447054594H1 2797 3403 44 70879623V1 2807 3487 44 5274874H1 2829 3072 4470007727D1 2843 3340 44 70010542D1 2843 3307 44 70010162D1 2843 3246 4470005864D1 2843 3198 44 70002001D1 2843 3074 44 70002333D1 2844 3415 4470011761D1 2844 3198 44 70001785D1 2849 3344 44 70007867D1 2874 3336 4470006872D1 2875 3344 44 70004362D1 2885 3284 44 70604116V1 1123 1734 442658395H1 3490 3738 44 70879732V1 3478 3911 44 g3429071 3484 3920 446317128H1 3442 3575 44 70879089V1 3455 3925 44 2661806T6 3469 3883 44700495H1 3477 3740 44 70608699V1 853 1342 44 70653541V1 904 1439 4470607650V1 918 1337 44 6938224H1 924 1338 44 70608866V1 964 1616 443776430H1 3217 3522 44 709518H1 3215 3449 44 70888779V1 3218 3398 44872814H1 3082 3286 44 5438843H1 3097 3403 44 70003362D1 3164 3424 4470004958D1 3165 3415 44 2527855H1 3178 3528 44 g1521303 3198 3655 44g1517127 3198 3698 44 2414483H1 3218 3454 44 70010299D1 3248 3632 4470005831D1 3338 3877 44 70003405D1 3101 3415 44 70007838D1 3099 3382 444880465H1 3100 3351 44 70012577D1 3107 3637 44 1320150H1 3127 3364 4470008556D1 3132 3440 44 4181419H1 1 167 44 6779195J1 66 705 44 113399R6430 794 44 4507995F6 435 610 44 4507995H1 436 607 44 6831490H1 443 63544 6831490J1 443 635 44 70604944V1 690 1146 44 70607511V1 785 1414 446454789H1 1287 1795 44 70603538V1 1322 1922 44 684735H1 1352 1601 4470607606V1 1355 1770 44 70603837V1 1402 1982 44 70006129D1 3099 3637 453386984H1 1 235 45 3087717H1 1 207 45 4832592H1 11 232 45 3750644H1 15214 45 3350574H1 18 296 45 3150464H1 24 307 45 3381160H1 29 281 453092918H1 38 363 45 3092958H1 38 329 45 1524230H1 43 257 45 3384786H1 92329 45 6055559H1 174 688 45 6055841H1 174 688 45 4509676H1 259 437 453081417H1 405 589 45 2952165H1 422 670 45 70874349V1 542 987

TABLE 5 SEQ ID NO: Template ID Tissue Distribution 1LG:977683.1:2000FEB18 Nervous System - 21%, Skin - 19%, EmbryonicStructures - 11% 2 LG:893050.1:2000FEB18 Digestive System - 40%, Hemicand Immune System - 40%, Nervous System - 20% 3 LG:980153.1:2000FEB18Nervous System - 16%, Urinary Tract - 12%, Skin - 12% 4LG:350398.1:2000FEB18 Digestive System - 50%, Hemic and Immune System -50% 5 LG:475551.1:2000FEB18 Skin - 35%, Hemic and Immune System - 19%,Digestive System - 11% 6 LG:481407.2:2000FEB18 widely distributed 7LI:443580.1:2000FEB01 Unclassified/Mixed - 60%, Connective Tissue - 17%,Endocrine System - 13% 8 LI:803015.1:2000FEB01 Urinary Tract - 63%,Respiratory System - 38% 9 LG:027410.3:2000MAY19 Respiratory System -100% 10 LG:171377.1:2000MAY19 Unclassified/Mixed - 74%, FemaleGenitalia - 13%, Cardiovascular System - 10% 11 LG:352559.1:2000MAY19Unclassified/Mixed - 71%, Digestive System - 29% 12LG:247384.1:2000MAY19 Stomatognathic System - 39%, MusculoskeletalSystem - 28%, Cardiovascular System - 19% 13 LG:403872.1:2000MAY19Nervous System - 40%, Embryonic Structures - 23%, Urinary Tract - 14% 14LG:1135213.1:2000MAY19 Embryonic Structures - 24%, CardiovascularSystem - 20%, Unclassified/Mixed - 13% 15 LG:474284.2:2000MAY19Unclassified/Mixed - 14% 16 LG:342147.1:2000MAY19 Pancreas - 21%, MaleGenitalia - 19%, Female Genitalia - 17%, Urinary Tract - 17% 17LG:1097300.1:2000MAY19 Endocrine System - 25%, Skin - 18%,Unclassified/Mixed - 13% 18 LG:444850.9:2000MAY19 Digestive System -28%, Connective Tissue - 20%, Exocrine Glands - 10% 19LG:402231.6:2000MAY19 Endocrine System - 23%, Hemic and Immune System -23%, Digestive System - 18% 20 LG:1076157.1:2000MAY19 EmbryonicStructures - 50%, Endocrine System - 28%, Respiratory System - 17% 21LG:1083142.1:2000MAY19 Germ Cells - 84% 22 LG:1083264.1:2000MAY19Liver - 52%, Connective Tissue - 33% 23 LG:350793.2:2000MAY19 SenseOrgans - 25%, Connective Tissue - 14% 24 LG:408751.3:2000MAY19 NervousSystem - 39%, Sense Organs - 39% 25 LI:336120.1:2000MAY01 NervousSystem - 24%, Respiratory System - 22%, Endocrine System - 18% 26LI:234104.2:2000MAY01 Female Genitalia - 21%, Unclassified/Mixed - 17%,Nervous System - 12% 27 LI:450887.1:2000MAY01 Nervous System - 100% 28LI:119992.3:2000MAY01 Embryonic Structures - 10% 29LI:197241.2:2000MAY01 Connective Tissue - 26%, Endocrine System - 12% 30LI:406860.20:2000MAY01 Digestive System - 100% 31 LI:142384.1:2000MAY01Connective Tissue - 44%, Germ Cells - 34% 32 LI:895427.1:2000MAY01Cardiovascular System - 20%, Urinary Tract - 14%, Skin - 13% 33LI:757439.1:2000MAY01 Digestive System - 18%, Embryonic Structures -13%, Sense Organs - 12% 34 LI:1144066.1:2000MAY01 CardiovascularSystem - 59%, Exocrine Glands - 25% 35 LI:243660.4:2000MAY01 Pancreas -63% 36 LI:334386.1:2000MAY01 Exocrine Glands - 17%, Male Genitalia -16%, Musculoskeletal System - 13% 37 LI:347572.1:2000MAY01 DigestiveSystem - 30%, Digestive System - 23%, Respiratory System - 17% 38LI:817314.1:2000MAY01 Unclassified/Mixed - 55%, Male Genitalia - 26%,Female Genitalia - 11% 39 LI:000290.1:2000MAY01 Female Genitalia - 54%40 LI:023518.3:2000MAY01 Urinary Tract - 50%, Musculoskeletal System -27%, Hemic and Immune System - 23% 41 LI:1084246.1:2000MAY01 SenseOrgans - 72% 42 LI:1165828.1:2000MAY01 Musculoskeletal System - 19%,Germ Cells - 18%, Nervous System - 14% 43 LI:007302.1:2000MAY01Connective Tissue - 29%, Respiratory System - 21%, Hemic and ImmuneSystem - 18% 44 LI:236386.4:2000MAY01 Skin - 30%, Female Genitalia - 11%45 LI:252904.5:2000MAY01 Exocrine Glands - 20%, Nervous System - 16%,Endocrine System - 13%

TABLE 6 SEQ ID Probability NO: Frame Length Start Stop GI Number scoreAnnotation 46 3 263 27 815 g10764778 1e−131 phosphoinositol3-phosphate-binding protein-2 [Homo sapiens] g10045840 1e−58 TPC2[unidentified] g4589582 2e−28 KIAA0969 protein [Homo sapiens] 47 1 21710 660 g6634025 1e−81 KIAA0379 protein [Homo sapiens] g6453538 6e−77hypothetical protein [Homo sapiens] g4803678 7e−29 ankyrin (brank-2)[Homo sapiens] 48 1 716 613 2760 g7243215 0.0 KIAA1417 protein [Homosapiens] g7263990 0.0 dJ93K22.1 (novel protein (contains DKFZP564B116))[Homo sapiens] g7302944 5e−57 CG8060 gene product [Drosophilamelanogaster] 49 3 107 60 380 50 3 645 3 1937 g4826478 0.0 dJ37E16.2(SH3-domain binding protein 1) [Homo sapiens] g861029 0.0 SH3 domainbinding protein [Mus musculus] g7018521 0.0 hypothetical protein [Homosapiens] 51 3 177 93 623 g6119546 1e−45 hypothetical protein;114721-113936 [Arabidopsis thaliana] g6522593 3e−10 putative RNA bindingprotein [Arabidopsis thaliana] g950424 4e−10 splicing factor,arginine/serine-rich 7 [Homo sapiens] 52 1 217 79 729 g4589566 3e−34KIAA0961 protein [Homo sapiens] g3970712 3e−26 zinc finger protein 10[Homo sapiens] g7630121 8e−25 zinc finger protein 92 [Mus musculus] 53 3151 3 455 g5262560 2e−35 hypothetical protein [Homo sapiens] g104348561e−29 unnamed protein product [Homo sapiens] g930123 9e−27 zinc fingerprotein (583 AA) [Homo sapiens] 54 3 193 3 581 g10438267 1e−74 unnamedprotein product [Homo sapiens] g7290756 8e−16 CG4532 gene product[Drosophila melanogaster] g5705877 8e−10 POD-1 [Caenorhabditis elegans]55 3 282 3 848 g3077703 1e−111 mitsugumin29 [Oryctolagus cuniculus]g3461888 1e−108 mitsugumin29 [Mus musculus] g3761107 1e−108 mitsugumin29[Mus musculus] 56 2 211 2 634 g7243243 2e−44 KIAA1431 protein [Homosapiens] g4567179 2e−43 BC37295_1 [Homo sapiens] g3445181 1e−41 R31665_2[Homo sapiens] 57 2 366 83 1180 g9945010 1e−120 RING-finger protein MURF[Mus musculus] g9929937 5e−92 hypothetical protein [Macaca fascicularis]g10439844 1e−36 unnamed protein product [Homo sapiens] 58 3 326 354 1331g7020303 0.0 unnamed protein product [Homo sapiens] g10434892 3e−79unnamed protein product [Homo sapiens] g6683707 2e−31 KIAA0455 protein[Homo sapiens] 59 1 156 70 537 g6692607 2e−69 MGA protein [Mus musculus]g5931585 9e−47 T-box family member; T-box domain [Cynops pyrrhogaster]g4049463 3e−16 transcription factor TBX6 [Homo sapiens] 60 2 262 2391024 g1488047 7e−12 RING finger protein [Xenopus laevis] g3916727 1e−11estrogen-responsive B box protein [Homo sapiens] g401763 1e−11ataxia-telangiectasia group D-associated protein [Homo sapiens] 61 3 132138 533 62 2 167 2 502 g2078531 2e−71 Mlark [Mus musculus] g20785292e−70 Hlark [Homo sapiens] g1149523 8e−57 Neosin [Mus musculus] 63 1 570160 1869 g183002 0.0 guanylate binding protein isoform I [Homo sapiens]g829177 0.0 guanylate binding protein isoform II [Homo sapiens] g70233320.0 unnamed protein product [Homo sapiens] 64 3 168 3 506 g7020737 2e−89unnamed protein product [Homo sapiens] g8920240 2e−89 AK000559hypothetical protein, similar to (U06944) PRAJA1 [Mus musculus] [Homosapiens] g2979531 2e−51 R33683_3 [Homo sapiens] 65 3 246 57 794 g52625603e−65 hypothetical protein [Homo sapiens] g10434856 4e−64 unnamedprotein product [Homo sapiens] g930123 7e−56 zinc finger protein (583AA) [Homo sapiens] 66 3 120 51 410 g4589566 2e−23 KIAA0961 protein [Homosapiens] g456269 7e−22 zinc finger protein 30 [Mus musculus domesticus]g5080758 2e−20 BC331191_1 [Homo sapiens] 67 2 122 329 694 g100472977e−26 KIAA1611 protein [Homo sapiens] g8163824 2e−19 krueppel-like zincfinger protein HZF2 [Homo sapiens] g3329372 6e−19 DNA-binding protein[Homo sapiens] 68 3 428 132 1415 g6094684 0.0 similar to Kelch proteins;similar to BAA77027 (PID: g4650844) [Homo sapiens] g7242973 0.0 KIAA1309protein [Homo sapiens] g7243089 0.0 KIAA1354 protein [Homo sapiens] 69 2307 2 922 g8671168 1e−135 hypothetical protein [Homo sapiens] g88860251e−135 collapsin response mediator protein-5 [Homo sapiens] g86713601e−131 Ulip-like protein [Rattus norvegicus] 70 1 198 856 1449 g18640851e−103 glypican-5 [Homo sapiens] g3015542 1e−103 glypican-5 [Homosapiens] g205800 7e−38 intestinal protein OCI-5 [Rattus norvegicus] 71 1227 511 1191 g1155088 1e−06 zyxin [Homo sapiens] g1545954 1e−06 zyxin[Homo sapiens] g576623 2e−06 ESP-2 [Homo sapiens] 72 3 122 3 368g7629994 4e−41 60S RIBOSOMAL PROTEIN L36 homolog [Arabidopsis thaliana]g3236242 5e−40 60S ribosomal protein L36 [Arabidopsis thaliana]g11908070 5e−40 60S ribosomal protein-like protein [Arabidopsisthaliana] 73 2 209 500 1126 g10435614 1e−113 unnamed protein product[Homo sapiens] g7243089 1e−113 KIAA1354 protein [Homo sapiens] g72429731e−107 KIAA1309 protein [Homo sapiens] 74 1 312 961 1896 g7243215 1e−157KIAA1417 protein [Homo sapiens] g7263990 1e−157 dJ93K22.1 (novel protein(contains DKFZP564B116)) [Homo sapiens] g7302944 3e−17 CG8060 geneproduct [Drosophila melanogaster] 75 3 190 3 572 g10435919 6e−69 unnamedprotein product [Homo sapiens] g3327128 3e−33 KIAA0657 protein [Homosapiens] g10436504 4e−09 unnamed protein product [Homo sapiens] 76 3 2953 887 g10436290 1e−105 unnamed protein product [Homo sapiens] g104360026e−99 unnamed protein product [Homo sapiens] g8489831 2e−27ubiquitin-conjugating BIR-domain enzyme APOLLON [Homo sapiens] 77 2 288374 1237 g3184264 5e−94 F02569_2 [Homo sapiens] g10435546 5e−84 unnamedprotein product [Homo sapiens] g6653742 4e−54 7h3 protein [Homo sapiens]78 1 294 97 978 g7670362 1e−106 unnamed protein product [Mus musculus]g6175860 4e−15 g1-related zinc finger protein [Mus musculus] g63305551e−13 KIAA1214 protein [Homo sapiens] 79 3 196 3 590 g3513300 3e−65F16601_1, partial CDS [Homo sapiens] g3882281 3e−50 KIAA0780 protein[Homo sapiens] g10567164 4e−50 gene amplified in squamous cellcarcinoma-1 [Homo sapiens] 80 3 745 285 2519 g2224553 0.0 KIAA0306 [Homosapiens] g4210501 0.0 BC85722_1 [Homo sapiens] g10728201 3e−20 CG2779gene product [Drosophila melanogaster] 81 3 256 507 1274 g6330617 1e−132KIAA1223 protein [Homo sapiens] g7301689 2e−72 CG10011 gene product[Drosophila melanogaster] g4803678 2e−33 ankyrin (brank-2) [Homosapiens] 82 1 235 841 1545 g9802433 2e−76 ACE-related carboxypeptidaseACE2 [Homo sapiens] g5817160 2e−76 hypothetical protein [Homo sapiens]g11876766 2e−76 unnamed protein product [Homo sapiens] 83 1 617 229 2079g6665594 0.0 trp-related protein 4 truncated variant delta [Homosapiens] g6665592 0.0 trp-related protein 4 truncated variant beta [Homosapiens] g6665590 0.0 trp-related protein 4 [Homo sapiens] 84 3 293 7351613 g7242977 1e−143 KIAA1311 protein [Homo sapiens] g912755 2e−15B0336.3 gene product [Caenorhabditis elegans] g7298595 8e−12 CG10084gene product [Drosophila melanogaster] 85 3 276 30 857 g3955100 2e−74vacuolar adenosine triphosphatase subunit D [Mus musculus] g12262352e−74 Ac39/physophilin [Mus musculus] g736727 2e−74 32 kd accessoryprotein [Bos taurus] 86 3 355 1392 2456 g5457043 0.0 protocadherin beta4 [Homo sapiens] g11142065 0.0 protocadherin beta 9 [Homo sapiens]g8926617 0.0 protocadherin 3H [Homo sapiens] 87 2 745 716 2950 g54570230.0 protocadherin alpha 9 short form protein [Homo sapiens] g3540157 0.0KIAA0345-like 5 [Homo sapiens] g2224631 0.0 KIAA0345 [Homo sapiens] 88 2781 50 2392 g5006248 0.0 TLR6 [Homo sapiens] g11596326 0.0 toll-likereceptor 6 [Mus musculus] g5006250 0.0 TLR6 [Mus musculus] 89 2 293 13132191 g6164628 2e−27 SH3 and PX domain-containing protein SH3PX1 [Homosapiens] g5327052 2e−27 dJ403L10.1 (SNX9 (Sorting Nexin 9)) [Homosapiens] g4689258 2e−27 sorting nexin 9 [Homo sapiens] 90 1 241 214 936g7022971 1e−62 unnamed protein product [Homo sapiens] g3882311 4e−15KIAA0795 protein [Homo sapiens] g4539520 4e−14 dA22D12.1 (novel proteinsimilar to Drosophila Kelch (Ring Canal protein, KEL) and a heterogenousset of other types of proteins) [Homo sapiens]

TABLE 7 Parameter Program Description Reference Threshold ABI A programthat removes vector sequences and Applied Biosystems, Foster City, CA.FACTURA masks ambiguous bases in nucleic acid sequences. ABI/ A FastData Finder useful in comparing and Applied Biosystems, Foster City, CA;Mismatch <50% PARACEL FDF annotating amino acid or nucleic acid ParacelInc., Pasadena, CA. sequences. ABI A program that assembles nucleicApplied Biosystems, Foster City, CA. AutoAssembler acid sequences. BLASTA Basic Local Alignment Search Tool useful Altschul, S. F. et al. (1990)J. Mol. Biol. ESTs: Probability in sequence similarity search for aminoacid 215: 403-410; Altschul, S. F. et al. (1997) value = 1.0E−8 or lessand nucleic acid sequences. BLAST includes five Nucleic Acids Res. 25:3389-3402. Full Length sequences: functions: blastp, blastn, blastx,tblastn, Probability value = and tblastx. 1.0E−10 or less FASTA APearson and Lipman algorithm that Pearson, W. R. and D. J. Lipman ESTs:fasta E searches for similarity between a query (1988) Proc. Natl. AcadSci. USA 85: value = 1.06E−6 sequence and a group of sequences of thesame 2444-2448; Pearson, W. R. (1990) Methods Assembled ESTs: fastatype. FASTA comprises as least five functions: Enzymol. 183: 63-98; andSmith, T. F. Identity = 95% or greater fasta, tfasta, fastx, tfastx, andssearch. and M. S. Waterman (1981) Adv. Appl. Math. and Match length =200 2: 482-489. bases or E value = 1.0E−8 or less greater; fastx FullLength sequences: fastx score =100 or greater BLIMPS A BLocks IMProvedSearcher that matches Henikoff, S. and J. G. Henikoff Probability value= a sequence against those in BLOCKS, (1991) Nucleic Acids Res. 19:6565-6572; 1.0E−3 or less PRINTS, DOMO, PRODOM, and PFAM databasesHenikoff, J. G. and S. Henikoff (1996) to search for gene families,sequence homology, Methods Enzymol. 266: 88-105; and Attwood, andstructural fingerprint regions. T. K. et al. (1997) J. Chem. Inf.Comput. Sci. 37: 417-424. HMMER An algorithm for searching a queryKrogh, A. et al. (1994) J. Mol. Biol., PFAM hits: sequence againsthidden Markov model 235: 1501-1531; Sonnhammer, Probability value =(HMM)-based databases of protein family consensus E. L. L. et al. (1988)Nucleic Acids Res. 26: 1.0E−3 or less Signal sequences, such as PFAM.320-322; Durbin, R. et al. (1998) Our World peptide hits: Score = 0View, in a Nutshell, Cambridge Univ. Press, or greater pp. 1-350.ProfileScan An algorithm that searches for structural Gribskov, M. etal. (1988) CABIOS 4: 61-66; Normalized quality and sequence motifs inprotein sequences that Gribskov, M. et al. (1989) Methods Enzymol. score≧ GCG- specified match sequence patterns defined in Prosite. 183:146-159; Bairoch, A. et al. (1997) “HIGH” value for that Nucleic AcidsRes. 25: 217-221. particular Prosite motif. Generally, score = 1.4-2.1.Phred A base-calling algorithm that examines Ewing, B. et al. (1998)Genome Res. automated sequencer traces with high sensitivity 8: 175-185;Ewing, B. and and probability. P. Green (1998) Genome Res. 8: 186-194.Phrap A Phils Revised Assembly Program including Smith, T. F. and M. S.Waterman (1981) Score = 120 or greater; SWAT and CrossMatch, programsbased on Adv. Appl. Math. 2: 482-489; Smith, Match length = 56 orefficient implementation of the Smith-Waterman T. F. and M. S. Waterman(1981) J. greater algorithm, useful in searching sequence homology Mol.Biol. 147: 195-197; and Green, P., and assembling DNA sequences.University of Washington, Seattle, WA. Consed A graphical tool forviewing and editing Phrap Gordon, D. et al. (1998) Genome Res. 8:195-202. assemblies. SPScan A weight matrix analysis program thatNielson, H. et al. (1997) Protein Engineering Score = 3.5 or greaterscans protein sequences for the presence of 10: 1-6; Claverie, J. M. andS. Audic (1997) secretory signal peptides. CABIOS 12: 431-439. TMAP Aprogram that uses weight matrices to Persson, B. and P. Argos (1994)delineate transmembrane segments on protein J. Mol. Biol. 237: 182-192;Persson, B. and sequences and determine orientation. P. Argos (1996)Protein Sci. 5: 363-371. TMHMMER A program that uses a hidden MarkovSonnhammer, E. L. et al. (1998) Proc. model (HMM) to delineatetransmembrane Sixth Intl. Conf. on Intelligent Systems for segments onprotein sequences and Mol. Biol., Glasgow et al., eds., The Am.determine orientation. Assoc. for Artificial Intelligence Press, MenloPark, CA, pp. 175-182. Motifs A program that searches amino acidBairoch, A. et al. (1997) Nucleic Acids sequences for patterns thatmatched those Res. 25: 217-221; Wisconsin Package defined in Prosite.Program Manual, version 9, page M51-59, Genetics Computer Group,Madison, WI.

1. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of: a) a polynucleotide sequence selected from the group consisting of SEQ ID NO:1-45, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO:1-45, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d).
 2. An isolated polynucleotide of claim 1, comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:1-45.
 3. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim
 1. 4. A composition for the detection of expression of disease detection and treatment molecule polynucleotides comprising at least one of the polynucleotides of claim 1 and a detectable label.
 5. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 1, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
 6. A method for detecting a target polynucleotide in a sample, said target polynucleotide comprising a sequence of a polynucleotide of claim 1, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.
 7. A method of claim 5, wherein the probe comprises at least 30 contiguous nucleotides.
 8. A method of claim 5, wherein the probe comprises at least 60 contiguous nucleotides.
 9. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim
 1. 10. A cell transformed with a recombinant polynucleotide of claim
 9. 11. A transgenic organism comprising a recombinant polynucleotide of claim
 9. 12. A method for producing a disease detection and treatment molecule polypeptide, the method comprising: a) culturing a cell under conditions suitable for expression of the disease detection and treatment molecule polypeptide, wherein said cell is transformed with a recombinant polynucleotide of claim 9, and b) recovering the disease detection and treatment molecule polypeptide so expressed.
 13. A purified disease detection and treatment molecule polypeptide (MDDT) encoded by at least one of the polynucleotides of claim
 2. 14. An isolated antibody which specifically binds to a disease detection and treatment molecule polypeptide of claim
 13. 15. A method of identifying a test compound which specifically binds to the disease detection and treatment molecule polypeptide of claim 13, the method comprising the steps of: a) providing a test compound; b) combining the disease detection and treatment molecule polypeptide with the test compound for a sufficient time and under suitable conditions for binding; and c) detecting binding of the disease detection and treatment molecule polypeptide to the test compound, thereby identifying the test compound which specifically binds the disease detection and treatment molecule polypeptide.
 16. A microarray wherein at least one element of the microarray is a polynucleotide of claim
 3. 17. A method for generating a transcript image of a sample which contains polynucleotides, the method comprising the steps of: a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray of claim 16 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.
 18. A method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence of claim 1, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
 19. A method for assessing toxicity of a test compound, said method comprising: a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 1 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 1 or fragment thereof; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
 20. An array comprising different nucleotide molecules affixed in distinct physical locations on solid substrate, wherein at least one of said nucleotide molecules comprises a first oligonucleotide or polynucleotide sequence specifically hybridizable with at least 30 contiguous nucleotides of a target polynucleotide, said target polynucleotide having a sequence of claim
 1. 21. An array of claim 20, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.
 22. An array of claim 20, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide
 23. An array of claim 20, which is a microarray.
 24. An array of claim 20, further comprising said target polynucleotide hybridized to said first oligonucleotide or polynucleotide.
 25. An array of claim 20, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.
 26. An array of claim 20, wherein each distinct physical location on the substrate contains multiple nucleotide molecules having the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another physical location on the substrate.
 27. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: a) an amino acid sequence selected from the group consisting of SEQ ID NO:46-90, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:46-90, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:46-90, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:46-90.
 28. An isolated polynucleotide encoding a polypeptide of claim
 13. 29. An isolated polynucleotide encoding a polypeptide of claim
 27. 30. A pharmaceutical composition comprising an effective amount of a polypeptide of claim 13 and a pharmaceutically acceptable excipient.
 31. A pharmaceutical composition comprising an effective amount of a polypeptide of claim 27 and a pharmaceutically acceptable excipient.
 32. A composition of claim 30, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:46-90.
 33. A composition of claim 31, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:46-90.
 34. A method of screening for a compound that specifically binds to the polypeptide of claim 13, the method comprising: a) combining the polypeptide of claim 13 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 13 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim
 13. 35. A method of screening for a compound that specifically binds to the polypeptide of claim 27, the method comprising: a) combining the polypeptide of claim 27 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 27 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim
 27. 36. A method of screening for a compound that modulates the activity of the polypeptide of claim 13, the method comprising: a) combining the polypeptide of claim 13 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 13, b) assessing the activity of the polypeptide of claim 13 in the presence of the test compound, and


48. A monoclonal antibody produced by a method of claim
 47. 49. A composition comprising the antibody of claim 48 and a suitable carrier.
 50. The antibody of claim 14, wherein the antibody is produced by screening a Fab expression library.
 51. The antibody of claim 14, wherein the antibody is produced by screening a recombinant immunoglobulin library.
 52. A method of detecting a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:46-90 in a sample, the method comprising: a) incubating the antibody of claim 14 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:46-90 in the sample.
 53. A method of purifying a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:46-90 from a sample, the method comprising: a) incubating the antibody of claim 14 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:46-90. 